HK40112929A - Processes for generating til products using pd-1 talen knockdown - Google Patents

Abstract

The present invention provides improved methods for expanding TILs and producing therapeutic populations of TILs, including methods for gene-editing at least a portion of the TILs to enhance their therapeutic efficacy. The methods lead to improved efficacy, improved phenotype, and increased metabolic health of the TILs in a shorter time period, while allowing for reduced microbial contamination as well as decreased costs. Such TILs find use in therapeutic treatment regimens.

Description

Methods for generating TIL products using PD-1 TALEN gene knockdown

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 63/242,373, filed September 9, 2021; U.S. Provisional Application No. 63/287,670, filed September 9, 2021; U.S. Provisional Application No. 63/322,190, filed March 21, 2022; U.S. Provisional Application No. 63/354,605, filed June 22, 2022; and U.S. Provisional Application No. 63/394,248, filed August 1, 2022, the disclosures of which are incorporated herein by reference in their entirety.

Background of the Invention

Using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) to treat large, refractory cancers represents an effective approach for treating patients with poor prognoses. (Gattinoni et al., Nat. Rev. Immunol. 2006, 6, 383-393). TILs are predominantly T-cells, and IL-2-based TIL expansion followed by a “rapid expansion process” (REP) is the preferred method for TIL expansion due to its speed and efficiency. (Dudley et al., Science 2002, 298, 850-54; Dudley et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. Immunother. 2003, 26, 332-42.) Numerous approaches have been explored to improve the response of melanoma to TIL therapy and to extend TIL therapy to other tumor types, but success has been limited and the field remains challenging. (Goff et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg et al., Clin. Cancer Res. 2011, 17, 4550-57.) Combination studies with single immune checkpoint inhibitors have also been described, but further investigation is underway and additional treatment options are needed (Kverneland et al., Oncotarget, 2020, 11(22), 2092-2105).

Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, aseptic issues, and other factors described herein, severely restricting their therapeutic potential for patients refractory to checkpoint inhibitor therapy. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes, suitable for treating patients with few or no viable treatment options. This invention addresses this need by providing a shortened manufacturing process for generating TILs.

The present invention provides improved and/or shortened processes and methods for amplifying TILs and generating therapeutic TIL populations, including methods for gene editing at least a portion of the therapeutic TIL population to enhance its therapeutic efficacy.

Summary of the Invention

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing of at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, the culturing or rapid second expansion of the fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the fourth TIL population into multiple subcultures; each subculture being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs or a therapeutic TIL population.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the activation of the second TIL population is performed using anti-CD3 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using OKT-3.

In some implementations, the step of activating the second TIL population is performed using 300 ng/mL of OKT-3.

In some implementations, the step of activating the second TIL population is performed using anti-CD3 agonist and anti-CD28 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using TransAct.

In some implementations, the step of activating the second TIL population is performed using TransAct diluted at 1:10, 1:17.5, or 1:100.

In some implementations, the step of activating the second TIL population takes about 2 days.

In some implementations, the step of activating the second TIL population takes about 3 days.

In some implementations, the step of activating the second TIL population takes approximately 4 days.

In some implementations, the step of activating the second TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the fourth TIL population takes about 8 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 9 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the step of culturing the fourth TIL population takes about 10 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A second TIL population was produced by culturing a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium for about 3–9 days in a first cell culture medium containing IL-2 and OKT-3;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some implementations, the step of culturing or rapidly expanding the third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the third TIL population into multiple subcultures; each of the subcultures being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the third TIL population takes about 8 days.

In some implementations, the step of culturing the third TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes about 8-9 days.

In some implementations, the step of culturing the third TIL population takes about 10 days.

In some implementations, the step of culturing the third TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, the step of culturing or initially amplifying the first TIL population in a first culture medium also includes anti-CD3 and anti-CD28 beads or antibodies.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population for about 3 days in a first cell culture medium containing IL-2 and anti-CD3 and anti-CD28 beads or antibodies, followed by culturing the first TIL population for 2-4 days in a cell culture medium containing IL-2 and OKT-3.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of at least two gene editors is transferred independently of the transfer of any other gene editor during the electroporation step.

In some implementations, the electroporation step also includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing IL-2 and/or IL-15.

In some embodiments, the first and second resting periods comprise incubating a third or fourth TIL population in a second cell culture medium containing 300 IU/mL, 1000 IU/mL, or 6000 IU/mL of IL-2.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing 15 ng/mL IL-15.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first and second resting periods comprise incubating a third or fourth TIL population at approximately 25, 28, 30, 32, 35, or 37°C with approximately 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some implementations, the electroporation step is preceded by washing a second or third TIL population in a cell pore buffer.

In some embodiments, the at least one gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates PD-1 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CTLA-4 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates LAG-3 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CISH expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CBL-B expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CTLA-4.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and TIGIT.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CISH and CBL-B.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, the tumor tissue is processed into multiple tumor fragments.

In some implementations, the tumor fragment is added to the closed system.

In some embodiments, 150 or fewer fragments, 100 or fewer fragments, or 50 or fewer fragments are added to the closed system. 167. A gene-edited tumor-infiltrating lymphocyte (TIL) population comprising an expanded TIL population, wherein the expression of at least one protein is regulated by a gene editor transferred to at least a portion of the expanded TIL population.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one protein is PD-1.

In some embodiments, the at least one protein is CTLA-4.

In some embodiments, the at least one protein is LAG-3.

In some embodiments, the at least one protein is CISH.

In some embodiments, the at least one protein is CBL-B.

In some embodiments, the at least one protein is TIGIT.

In some embodiments, the expression of the at least two proteins is regulated by at least two gene editors transferred to at least a portion of the amplified TIL population, wherein the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of the first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of the second protein.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first TALE protein and the second TALE protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some implementations, the gene-edited TIL populations disclosed herein are created using the methods disclosed herein.

In some embodiments, this document provides a pharmaceutical composition comprising the gene-edited TIL population disclosed herein and a pharmaceutically acceptable vector.

In some implementations, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of the gene-edited TIL population disclosed herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) The tumor fragment is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step includes delivering a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of the TALEN system for inhibiting CTLA-4 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CBL-B and TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is about 1× ^10⁹ to about 1× ^10¹¹ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of the collected TIL population in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the patient has been given a therapeutically effective dose of the collected TIL population in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Attached Figure Description

Figure 1: Exemplary Gen 2 (Process 2A) diagram, which provides an overview of steps A through F.

Figures 2A to 2C: Process flow diagrams of one implementation of Gen 2 (process 2A) for TIL manufacturing.

Figure 3: A diagram showing one embodiment of an exemplary manufacturing process for cryopreserved TILs (approximately 22 days).

Figure 4: A diagram showing one implementation of Gen 2 (Process 2A, a 22-day process for TIL manufacturing).

Figure 5: Comparison table of steps A to F of an exemplary implementation of process 1C and Gen 2 (process 2A) for TIL manufacturing.

Figure 6: A detailed comparison of one implementation of Process 1C and one implementation of Gen 2 (Process 2A) for TIL manufacturing.

Figure 7: Exemplary Gen 3 type TIL manufacturing process.

Figures 8A through 8P: A) A comparison between an implementation of the 2A process (approximately 22 days) and a Gen 3 process (approximately 14 to 16 days) for TIL manufacturing. B) An exemplary Gen 3 process diagram providing an overview of steps A through F (approximately 14 to 16 days). C) Diagrams of three exemplary Gen 3 processes, outlining steps A through F (approximately 14 to 16 days) for each of the three process variations. D) An exemplary modified Gen 2-like process providing an overview of steps A through F (approximately 22 days). E-P) Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 9: Experimental flowchart providing comparability between Gen 2 (process 2A) and Gen 3 processes.

Figure 10: A comparison of various Gen 2 (Process 2A) and Gen 3.1 process implementation schemes.

Figure 11: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 12: An overview of the culture medium conditions for one implementation of the Gen 3 process (referred to as Gen 3.1).

Figure 13: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 14: A table comparing various features of the implementation schemes for the Gen 2 and Gen 3.0 processes.

Figure 15: A table showing the culture medium used in various implementation schemes of the described amplification process.

Figure 16: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 17: A schematic diagram of an exemplary implementation of a method for amplifying T cells from hematopoietic malignancies using the Gen 3 amplification platform.

Figure 18: Structures IA and IB are provided. The cylinder refers to an individual polypeptide-binding domain. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from, for example, 4-1BBL or antibodies binding to 4-1BB, which fold to form a trivalent protein. This trivalent protein is then linked to a second trivalent protein via an IgG1-Fc (including CH3 and CH2 domains), which in turn links the two trivalent proteins together via disulfide bonds (small elongated ellipses). This stabilizes the structure and provides an agonist capable of aggregating the intracellular signaling domains of six receptors with signaling proteins to form a signaling complex. The TNFRSF-binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys sequences.

Figure 19: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 20: Process overview of an exemplary implementation of the Gen 3.1 process (16-day process).

Figure 21: A schematic diagram of an exemplary implementation of the Gen 3.1 testing process (16-day to 17-day process).

Figure 22: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 23: Comparison table of exemplary Gen 2 and exemplary Gen 3 processes.

Figure 24: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day to 17-day process) preparation timeline.

Figure 25: A schematic diagram of an exemplary implementation of the Gen 3 process (14-day to 16-day process).

Figures 26A to 26B: Schematic diagrams of an exemplary implementation of the Gen 3 process (16-day process).

Figure 27: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 28: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 29: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 30: Components of the Gen 3 implementation scheme.

Figure 31: Comparison of Gen 3 implementation flowcharts (Gen 3.0, Gen 3.1 comparison, Gen 3.1 test).

Figure 32: Shows components of an exemplary implementation of the Gen 3 process (16- to 17-day process).

Figure 33: Acceptance Criteria Table.

Figure 34: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figure 35: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figures 36A to 36D: Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 37: A schematic diagram of an exemplary implementation of the process described in Example 12.

Figures 38A-38B: In vivo efficacy of PDCD-1KO TIL. A) PDCD-1KO efficiency as assessed by flow cytometry. B) hIL-2NOG mice with melanoma tumor cells transplanted via adoptive transfer using PDCD-1KO or TIL-mimicking mice (n=14 per treatment group). Anti-PD-1 antibody treatment combined with TIL-mimicking mice served as a control for PD-1/PD-L1 blockade. Statistical significance is indicated by *p<0.05, **p<0.01, and ****p<0.0001.

Figures 39A to 39E: TIL product analysis. A) Live cell dose, B) Purity, C) Identity, D) Potency, and E) PDCD-1KO efficiency of TIL products.

Figures 40A to 40B: TIL product analysis. A) TIL differentiation and B) TIL memory.

Figures 41A to 41B: Expression of activation and inhibition of related biomarkers on PDCD-1KO TIL.

Figures 42A to 42B: IL-2-independent proliferation assay of PDCD-1KO TIL products.

Figure 43: Summary of karyotype analysis results of PDCD-1KO TIL products.

Figures 44A to 44B: Cell viability (Figure 44A) and recovery fold (Figure 44B) before electroporation.

Figures 45A to 45B: Cell recovery fold (Figure 45A) and cell viability (Figure 45B) after electroporation.

Figures 46A to 46C: Gene knockout efficiency of CD3+ (Figure 46A), CD8+ (Figure 46B), and CD4+ (Figure 46C) cells.

Figures 47A to 47B: Cell recovery fold (Figure 47A) and cell viability (Figure 47B) after electroporation.

Figures 48A to 48B: Cell recovery fold (Figure 48A) and cell viability (Figure 48B) after electroporation when using 6000 IU/mL IL-2.

Figures 49A to 49B: Cell recovery fold (Figure 49A) and cell viability (Figure 49B) after electroporation under various conditions.

Figures 50A to 50C: Gene knockout efficiency of CD3+ (Figure 50A), CD8+ (Figure 50B), and CD4+ (Figure 50C) cells.

Figure 51: Cell viability before electroporation.

Figure 52: Cell resuscitation fold before electroporation.

Figures 53A to 53B: Cell recovery fold (Figure 53A) and cell viability (Figure 53B) after electroporation.

Figures 54A to 54C: Gene knockout efficiency of CD3+ (Figure 54A), CD8+ (Figure 54B), and CD4+ (Figure 54C) cells.

Figures 55A to 55B: Cell number (Figure 55A) and survival rate (Figure 55B) after various washing steps.

Figures 56A to 56B: Cell numbers after various rotation conditions using PBS washing (Figure 56A) or Cyto washing (Figure 56B).

Figures 57A to 57B: Cell viability after various rotation conditions using PBS washing (Figure 57A) or Cyto washing (Figure 57B).

Figures 58A to 58B: Total rotation comparison of cell number (Figure 58A) and total rotation comparison of cell survival rate (Figure 58B) after various rotation conditions.

Figure 59: Percentage of cell loss after total rotation under various rotation conditions.

Figures 60A to 60C: Percentage of cell loss and survival rate during electroporation, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation.

Figures 61A to 61C: Gene knockout efficiency of CD3+ (Figure 61A), CD8+ (Figure 61B), and CD4+ (Figure 61C) cells.

Figures 62A to 62B: Cell viability (Figure 62A) and fold expansion (Figure 62B) of the REP collection.

Figures 63A to 63B: Percentage of cell loss after electroporation (Figure 63A) and cell viability (Figure 63B).

Figures 64A to 64C: Gene knockout efficiency of CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells.

Figures 65A to 65B: Folding of REP collection (Figure 65A) and cell viability (Figure 65B).

Figures 66A to 66C: Cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C).

Figure 67: Percentage of growth after 3 days of rest.

Figures 68A to 68C: PD-1 gene knockout efficiency.

Figure 69: PDCD1 gene modification according to NGS.

Figures 70A to 70B: Distribution of the TCR Vβ isotype in the CD3+PD-1- subset in the main PD-1KO TIL product and NE TIL.

Figures 71A to 71B: PD-1KO TIL effect function as measured by MLR (Figure 71A) and multifunctionality (Figure 71B).

Figure 72: In vivo antitumor activity of M1152 PD-1KO TIL product.

Figures 73A to 73B: Durability of TALEN protein in autologous TILs as a function of time, measured by Western blot.

Figures 74A-F: Exemplary TIL manufacturing process.

Figures 75A-B: Schematic diagrams of the 1/2 phase study described in Example 22.

Figure 76: Summary of data described in Example 23.

Figures 77A-D: Results of the demonstration day experiment from Example 23.

Figures 78A-C: Results of neon experiment 1 from Example 23.

Figures 79A-C: Results of Xenon Experiment 1 from Example 23.

Figures 80A-B: Results of Xenon Experiment 3 from Example 23.

Figures 81A-C: Results of Xenon Experiment 4 from Example 23.

A brief explanation of sequence listings

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of moromumab.

SEQ ID NO:3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO:4 is the amino acid sequence of adefovir.

SEQ ID NO:5 is in IL-2 form.

SEQ ID NO:6 is the amino acid sequence of Nevo interleukin-α.

SEQ ID NO:7 is in IL-2 form.

SEQ ID NO:8 is a mucin domain polypeptide.

SEQ ID NO:9 is the amino acid sequence of recombinant human IL-4 protein.

SEQ ID NO:10 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO:11 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO:12 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO:13 is an IL-2 sequence.

SEQ ID NO:14 is the sequence of the IL-2 mutant protein.

SEQ ID NO:15 is the sequence of the IL-2 mutant protein.

SEQ ID NO:16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

SEQ ID NO:17 is HCDR2 of IgG.IL2R67A.H1.

SEQ ID NO:18 is HCDR3 of IgG.IL2R67A.H1.

SEQ ID NO:19 is HCDR1_IL-2kabat of IgG.IL2R67A.H1.

SEQ ID NO:20 is HCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:21 is HCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:22 is HCDR1_IL-2clothia of IgG.IL2R67A.H1.

SEQ ID NO:23 is HCDR2 clothia of IgG.IL2R67A.H1.

SEQ ID NO:24 is HCDR3 clothia of IgG.IL2R67A.H1.

SEQ ID NO:25 is HCDR1_IL-2IMGT of IgG.IL2R67A.H1.

SEQ ID NO:26 is HCDR2 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:27 is HCDR3 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:28 is the V _H chain of IgG.IL2R67A.H1.

SEQ ID NO:29 is the heavy chain of IgG.IL2R67A.H1.

SEQ ID NO:30 is the LCDR1 kabat of IgG.IL2R67A.H1.

SEQ ID NO:31 is the LCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:32 is the LCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:33 is the LCDR1 chothia of IgG.IL2R67A.H1.

SEQ ID NO:34 is the LCDR2 chothia of IgG.IL2R67A.H1.

SEQ ID NO:35 is the LCDR3 chothia of IgG.IL2R67A.H1.

SEQ ID NO:36 is the V _L chain.

SEQ ID NO:37 is a light chain.

SEQ ID NO:38 is a light chain.

SEQ ID NO:39 is a light chain.

SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

SEQ ID NO:41 is the amino acid sequence of mouse 4-1BB.

SEQ ID NO:42 is the heavy chain of utomilumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:43 is the light chain of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:44 is the heavy chain variable region (V _H ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:45 is the light chain variable region (V _L ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:46 is the heavy chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:47 is the heavy chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:48 is the heavy chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:49 is the light chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:50 is the light chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:51 is the light chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:52 is the heavy chain of urelumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:53 is the light chain of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:54 is the heavy chain variable region (VH) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:55 is the light chain variable region (VL) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:56 is the heavy chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:57 is the heavy chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:58 is the heavy chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:59 is the light chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:60 is the light chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:61 is the light chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:62 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:63 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:64 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:65 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:66 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:67 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:68 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:69 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:70 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:71 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:72 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:73 represents the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:74 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:75 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:76 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:77 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

SEQ ID NO:78 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO:79 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 1.

SEQ ID NO:80 is the light chain variable region (V _L ) of type 1 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:81 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 2.

SEQ ID NO:82 is the light chain variable region (V _L ) of type 2 of 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:83 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:84 is the light chain variable region (V _L ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:85 is the amino acid sequence of human OX40.

SEQ ID NO:86 is the amino acid sequence of mouse OX40.

SEQ ID NO:87 is the heavy chain of the OX40 agonist monoclonal antibody taboliziumab (MEDI-0562).

SEQ ID NO:88 is the light chain of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:89 is the heavy chain variable region (V _H ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:90 is the light chain variable region (V _L ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:91 is the heavy chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:92 is the heavy chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:93 is the heavy chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:94 is the light chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:95 is the light chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:96 is the light chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:99 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:100 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:107 is the heavy chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:108 is the light chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:109 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:110 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:117 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:118 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:119 is the heavy chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:120 is the heavy chain CDR2 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:122 is the light chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:125 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:126 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:130 is the light chain CDR1 of Hu106-222, an OX40 agonist monoclonal antibody.

SEQ ID NO:131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:133 is the amino acid sequence of the OX40 ligand (OX40L).

SEQ ID NO:134 is the soluble portion of the OX40L polypeptide.

SEQ ID NO:135 is an alternative soluble fraction of the OX40L polypeptide.

SEQ ID NO:136 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:137 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:138 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:139 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:140 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 021.

SEQ ID NO:141 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 021.

SEQ ID NO:142 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:143 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:144 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:145 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:146 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:147 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:148 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:149 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:150 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:151 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:152 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:153 is the heavy chain variable region ( _VH ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:154 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:155 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:156 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:157 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:160 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:161 is the amino acid sequence of the light chain variable region ( _VL ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:170 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:171 is the amino acid sequence of the light chain variable region (V _L ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:180 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:181 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:190 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:191 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:200 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:201 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:210 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:211 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:220 is the amino acid sequence of the heavy chain variable region (V _H ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:221 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4 inhibitor zeflimab.

SEQ ID NO:230 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:231 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:238 is an exemplary Clo05 l nuclease domain amino acid sequence.

SEQ ID NO:239 is an exemplary piggyBac(PB) transposase amino acid sequence.

SEQ ID NO:240 is an exemplary Sleeping Beauty transposase amino acid sequence.

SEQ ID NO:241 is an exemplary amino acid sequence of an overactive Sleeping Beauty (SB100X) transposase.

I. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and disclosures mentioned herein are incorporated herein by reference in their entirety.

As used herein, the terms "co-administration/co-administering," "administered in combination with/administering in combination with," "simultaneous," and "concurrent" encompass the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, such as multiple TILs) to a subject, such that both the active pharmaceutical ingredient and/or its metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of separate compositions and administration of a composition in which two agents are present are preferred.

The term "in vivo" refers to events that occur within the body of a subject.

The term "in vitro" refers to an event that occurs outside the subject's body. In vitro assays encompass cell-based assays using live or dead cells, and may also include cell-free assays that do not use intact cells.

The term "ex vivo" refers to an event involving the treatment or procedure performed on cells, tissues, and/or organs that have been removed from a subject's body. Appropriately, cells, tissues, and/or organs may be returned to the subject using surgical or therapeutic methods.

The term "rapid amplification" means that the number of antigen-specific TILs increases at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) within one week, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) within one week, or most preferably at least about 100-fold within one week. Several rapid amplification protocols are described herein.

In this article, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are TILs obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any expanded or proliferated population of TIL cells as discussed herein, including (but not limited to) main TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations may contain genetically modified TILs.

In this article, "cell population" (including TILs) refers to a large number of cells sharing common characteristics. Typically, the population size ranges from 1 × ^10⁶ to 1 × ^10¹⁰ cells, with different TIL populations containing varying numbers. For example, initial growth of primary TILs in the presence of IL-2 produces a main TIL population of approximately 1 × ^10⁸ cells. REP expansion is typically performed to provide a cell population of 1.5 × ^10⁹ to 1.5 × ^10¹⁰ cells for infusion.

In this document, “cryopreserved TIL” means TIL that has been processed and stored in the range of approximately -150°C to -60°C, whether primary, host, or amplified (REP TIL). General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, “cryopreserved TIL” may be distinguished from frozen tissue samples that can be used as a source of primary TIL.

In this article, “thawed cryopreserved TILs” means a population of TILs that were previously cryopreserved and then processed to be restored to room temperature or higher (including, but not limited to, cell culture temperatures or temperatures at which TILs can be administered to patients).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to reintroduce into patients with late-stage invasive solid tumors.

The term "cryopreservation media" refers to any culture medium that can be used for cryopreservation of cells. Such media may include those containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 media may be referred to by the trade name "CS10". CS10 media is a serum-free, animal-free medium containing DMSO. In some embodiments, CS10 media contains 10% DMSO.

The term "central memory T cells" refers to a subset of T cells in humans that are CD45R0+ and constitutively express CCR7 (CCR7 ^hi ) and CD62L (CD62 ^hi ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Upon TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are mainly found in the CD4 compartments of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

The term "effective memory T cells" refers to a subset of human or mammalian T cells, such as central memory T cells, which are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 ^lo ) and are heterogeneous or low in expression of CD62L (CD62L ^lo ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, upon antigen stimulation. Effector memory T cells are primarily found in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry a large amount of perforin.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, for example (but not limited to), closed G containers. Once a tumor segment is added to the closed system, the system remains closed to the external environment until the TIL is ready for administration to the patient.

As used herein, the terms “fragmenting,” “fragment,” and “fragmented” describe the process of destroying a tumor, including mechanical fragmentation methods such as crushing, slicing, splitting, and pulverizing tumor tissue, as well as any other methods used to destroy the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBL is separated from whole blood or apheresis products from a donor. In some embodiments, PBL is separated from whole blood or apheresis products from a donor by positively or negatively selecting for a T cell phenotype (e.g., CD3+CD45+ T cell phenotype).

The term "anti-CD3 antibody" refers to an antibody or a variant thereof that targets the CD3 receptor in the T-cell antigen receptor of mature T cells, such as a monoclonal antibody, and includes human, humanized, chimeric, murine, or mammalian antibodies. Anti-CD3 antibodies include OKT-3, also known as moromuzumab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof targeting the CD3 receptor in the T-cell antigen receptor of mature T cells, including human, humanized, chimeric, or murine antibodies, and including commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, San Diego, CA, USA) and moromumab or its variants, conserved amino acid substitutions, glycosylated forms, or biosimilars. The amino acid sequences of the heavy and light chains of moromumab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). Hybridomas capable of producing OKT-3 are deposited at the American Type Culture Collection and designated ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited at the European Collection of Authenticated Cell Cultures (ECACC) with catalog number 86022706.

Table 1. Amino acid sequence of moromuzumab (exemplary OKT-3 antibody)

The term "IL-2" (also referred to herein as "IL2") refers to a T-cell growth factor called interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in this invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses recombinant human IL-2, such as adefovir (PROLEUKIN, available from multiple suppliers, 22 million IU per single-use vial), as well as recombinant IL-2 forms supplied by CellGenix (CELLGRO GMP) of Portsmouth, NH, USA, or ProSpec-Tany TechnoGene Ltd. (catalog number CYT-209-b) of East Brunswick, NJ, USA, and other commercial equivalents from other suppliers. Adefovir (desalanyl-1, serine-125 human IL-2) is a non-glycosylated recombinant human IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of the adefovir suitable for use in this invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including the PEGylated IL-2 prodrug beempegaldesleukin (NKTR-214, as SEQ ID NO:4, PEGylated recombinant human IL-2 wherein an average of 6 lysine residues are substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluorene-9-yl)methoxy]carbonyl N ⁶ ), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO 2018/132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US2019/0275133 A1, the disclosure of which is incorporated herein by reference. The bepedelion interleukin (NKTR-214) and other PEGylated IL-2 molecules applicable to the present invention are described in U.S. Patent Application Publication No. US2014/0328791A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 applicable to the present invention are described in U.S. Patents Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations applicable to the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

In some embodiments, the IL-2 form suitable for use in this invention is THOR-707, available from Synthorx. The preparation and characteristics of THOR-707 and additional alternative forms of IL-2 suitable for use in this invention are described in U.S. Patent Application Publications Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for use in this invention is an interleukin-2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugate portion bound to the isolated and purified IL-2 polypeptide at amino acid positions selected from the following: K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the amino acid residues are numbered corresponding to SEQ ID NO: 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residues are mutated to cysteine. In some embodiments, the amino acid residues are mutated to lysine. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to non-natural amino acids. In some embodiments, the non-natural amino acids include N6-azidoethoxy-L-lysine (AzK), N6-propynylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, 3-methyl-phenylalanine, L-DOPA, fluorinated phenylalanine, Isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzoxy)-3-oxopropyl)amino)ethyl)selenoyl)propionic acid, 2-amino-3-(phenylselenoyl)propionic acid, or selenocysteine. In some embodiments, the IL-2 conjugate exhibits reduced affinity for the IL-2 receptor α (IL-2Rα) subunit relative to the wild-type IL-2 peptide. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% of the binding affinity to IL-2Rα. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 100, 200, 300, 500, 1000, or greater. In some embodiments, the conjugated moiety weakens or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugation portion comprises a water-soluble polymer. In some embodiments, each water-soluble polymer independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each water-soluble polymer independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises polydextrose, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the water-soluble polymers each independently comprise a polyamine. In some embodiments, the conjugated moiety comprises a protein. In some embodiments, an additional conjugated moiety comprises a protein. In some embodiments, the proteins each independently comprise albumin, transferrin, or transthyretin. In some embodiments, the proteins each independently comprise an Fc moiety. In some embodiments, the proteins each independently comprise the Fc moiety of IgG. In some embodiments, the conjugated moiety comprises a peptide. In some embodiments, an additional conjugated moiety comprises a peptide. In some embodiments, the peptides each independently comprise XTEN peptides, high-amino acid polymers rich in glycine (HAP), PAS peptides, elastin-like peptides (ELP), CTP peptides, or gelatin-like protein (GLK) polymers. In some embodiments, the isolated and purified IL-2 peptide is modified by glutamylation. In some embodiments, the conjugated moiety binds directly to the isolated and purified IL-2 peptide. In some embodiments, the conjugated moiety binds indirectly to the isolated and purified IL-2 peptide via a linker. In some embodiments, the linker comprises a homologous bifunctional linker. In some embodiments, the homodifunctional linker comprises Lomant's reagent, dithiobis(succinimide propionate) DSP, 3'3'-dithiobis(succinimide propionate sulfonyl succinimide) (DTSSP), succinimide disuccinate (DSS), succinimide disuccinate (BS), succinimide tartrate (DST), disulfonyl tartrate (sulfonyl DST), glycosylbis(succinimide succinate) ethylene ester (EGS), glutarate succinimide (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl hexamethylenediimide (DMA), dimethyl heptamethine (DMP), dimethyl octanediimide (DMS), dimethyl-3,3'-dithiobispropionide (DSP) DTBP), 1,4-bis(3'-(2'-pyridyldithio)propamido)butane (DPDPB), bis(cis-butenyldiimide)hexane (BMH), compounds containing aryl halides (DFDNB) (e.g., 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the connector comprises a heterogeneous bifunctional connector. In some embodiments, the heterogeneous bifunctional connector comprises 3-(2-pyridyldithio)propionic acid N-succinimide ester (sPDP), long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (LC-sPDP), water-soluble long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (sulfonyl-LC-sPDP), succinimide-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), or sulfosuccinimide-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate. (Sulfon-LC-sMPT), succinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sulfon-sMCC), m-cis-butenyldiimide benzoyl-N-hydroxysuccinimide (MBs), m-cis-butenyldiimide benzoyl-N-hydroxysulfosuccinimide (sulfon-MBs), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB) ) sulfosuccinimide aminobenzoate (sulfon-sIAB), succinimide-4-(p-cis-butenylimide-phenyl)butyrate (sMPB), sulfosuccinimide-4-(p-cis-butenylimide-phenyl)butyrate (sulfon-sMPB), N-(γ-cis-butenylimide-butyryloxy)succinimide (GMBs), N-(γ-cis-butenylimide-butyryloxy)sulfosuccinimide (sulfon-GMBs), 6-((iodoacetyl)amino)hexanoic acid succinimide (sIAX), 6-[6-(((iodoacetyl)amino)hexanoic acid succinimide Acetyl)amino)hexanoyl)amino]hexanoic acid succinimide ester (sIAXX), 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylic acid succinimide ester (sIAC), 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoic acid succinimide ester (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl reactive and thiohydrogen-reactive crosslinking agents, such as 4-(4-N-cis-butenyldiimide-phenyl)butyric acid hydrazide (MPBH), 4-(N-cis-butenyldiimide-methyl)cyclohexane- 1-Carboxy-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfon-NHs-AsA), sulfosuccinimide-(4-azidosalicylamido)hexanoate (sulfon-NHs-LC-AsA), sulfosuccinimide-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimide-4 -Azide benzoate (HsAB), N-hydroxysulfosuccinimide-4-azidobenzoate (sulfon-HsAB), N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfon-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimide-2-(m-azido-o-nitrobenzoamide)-ethyl-1,3' -Dithiopropionate (sAND), N-succinimide-4-(4-azidophenyl)1,3'-dithiopropionate (sADP), (4-azidophenyl)-1,3'-dithiopropionate N-sulfosuccinimide (sulfon-sADP), 4-(p-azidophenyl)butyrate sulfosuccinimide (sulfon-sAPB), 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate sulfosuccinimide (sAED), 7-azido-4-methylcoumarin-3-acetic acid sulfosuccinimide (sulfon-s... AMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleic anhydride group, optionally comprising a maleic anhydride hexanoyl (mc), succinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sulfon-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives thereof, or the like. In some embodiments, the conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, an additional conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in this invention is polyethylene glycol-modified as disclosed in U.S. Patent Application Publication US 2020/0181220 A1 and U.S. Patent Application Publication US 2020/0330601 A1. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion relative to one residue of SEQ ID NO:5. In some embodiments, the IL-2 form suitable for the present invention lacks the IL-2Rα linker but maintains normal binding to the intermediate affinity IL-2Rβ-γ signaling complex. In some embodiments, the IL-2 form suitable for the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5.

In some embodiments, the IL-2 form suitable for use in this invention is Nevor interleukin-α (also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes). Nevo interleukin-α, also known as the human interleukin-2 fragment (1-59) variant (Cys ¹²⁵ > Ser ⁵¹ ), is fused to the human interleukin-2 fragment (62-132) via a peptide linker ( ⁶⁰ GG ⁶¹ ), which is fused to the human interleukin-2 receptor α chain fragment (139-303) via a peptide linker ( ¹³³ GSGGGS ¹³⁸ ). It is produced in Chinese hamster ovary (CHO) cells and is glycosylated. The human interleukin-2 (IL-2) (75-133)-peptide [Cys ¹²⁵ (51) > Ser]-mutant (1-59) is fused to the human interleukin-2 (IL-2) (4-74)-peptide (62-132) via a _G2 peptide linker (60-61) and via GSG _3. The S-peptide linker (133-138) is fused to the human interleukin 2 receptor α chain (IL2R subunit α, IL2Rα, IL2RA) (1-165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, in glycoform α. The amino acid sequence of Nevo interleukin α is provided in SEQ ID NO:6. In some embodiments, Nevo interleukin α exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbers in SEQ ID NO:6), and glycosylation sites at the following positions: N187, N206, T212 (using the numbers in SEQ ID NO:6). The preparation and properties of Nevo interleukin-α, as well as additional alternative forms of IL-2 suitable for the present invention, are described in U.S. Patent Application Publication No. US2021/0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the IL-2 form suitable for the present invention has the amino acid sequence provided in SEQ ID NO:6 or its conserved amino acid substitutions. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner via a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity with IL-1Rα and having receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises an immunoglobulin comprising all or part of an Fc region, wherein the mucin domain polypeptide linker comprises an amino acid sequence of SEQ ID NO:8 or having at least 90% sequence identity with SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

Table 2. Amino acid sequence of interleukin.

In some embodiments, the IL-2 form suitable for use in this invention comprises an antibody cytokine transplantation protein comprising: a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some implementations, the IL-2 regimen comprises administering the antibody described in U.S. Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine transplantation protein comprises: a heavy chain variable region (VH) including complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region (VL) including LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into a CDR of the _VH or _VL , wherein the IL-2 molecule is a mutant protein, wherein the antibody cytokine transplantation protein preferentially amplifies T effector cells relative to regulatory T cells, and wherein the antibody further comprises an IgG heavy chain and an IgG light chain selected from the group consisting of: an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:38; an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:29; an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:29; and an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:38.

In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR1 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR2 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR3 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR1 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR2 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR3 of _VL , wherein the IL-2 molecule is a mutant protein.

The insertion of an IL-2 molecule can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence replaces all or part of the CDR sequence. The IL-2 molecule replacement can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. The IL-2 molecule replacement can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

In some embodiments, the IL-2 molecule is directly grafted into a CDR without a peptide linker, wherein there are no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted into a CDR with a peptide linker, wherein there are one or more additional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecule described herein is an IL-2 mutant protein. In some cases, the IL-2 mutant protein contains an R67A substitution. In some embodiments, the IL-2 mutant protein contains the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutant protein contains the amino acid sequence of Table 1 in U.S. Patent Application Publication No. US 2020/0270334A1, the disclosure of which is incorporated herein by reference.

In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine transplantation protein comprises HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine transplantation protein comprises a _VH region containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine transplantation protein includes a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a light chain containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a _VH region containing the amino acid sequence of SEQ ID NO:28, and a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:38, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain region containing the amino acid sequence of SEQ ID NO:38; and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 or variants, derivatives, or fragments thereof, or conserved amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with them, as described in U.S. Patent Application Publication No. 2020/0270334A1. In some embodiments, the antibody component of the antibody cytokine transplantation protein described herein comprises the immunoglobulin sequence, framework sequence, or CDR sequence of palizumab. In some embodiments, the serum half-life of the antibody cytokine transplantation protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to aldehyde interleukin or similar molecules). In some embodiments, the antibody cytokine transplantation protein described herein has a sequence as illustrated in Table 3.

Table 3: Sequence of exemplary palizumab antibody-IL-2 transplanted protein

The term “IL-4” (also referred to herein as “IL4”) refers to a cytokine known as interleukin-4, produced by Th2 T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and _IgG1 expression. Recombinant human IL-4 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-211) and ThermoFisher Scientific of Waltham, MA, USA (recombinant human IL-15 protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for this invention is provided in Table 2 (SEQ ID NO:9).

The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine called interleukin-7, which is available from stromal and epithelial cells as well as dendritic cells. (Fry and Mackall, Blood 2002, 99, 3892-904). IL-7 stimulates T cell development. IL-7 binds to the IL-7 receptor (a heterodimer composed of IL-7 receptor α and a common γ chain receptor), which belongs to a family of signaling pathways important for T cell development within the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-254) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for this invention is provided in Table 2 (SEQ ID NO: 10).

The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor called interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the contents of which are incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-230-b) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number 34-8159-82). The amino acid sequence of the recombinant human IL-15 suitable for use in this invention is provided in Table 2 (SEQ ID NO: 11).

The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the contents of which are incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-408-b) and Thermo Fisher Scientific of Waltham, Massachusetts (recombinant human IL-21 protein, catalog number 14-8219-80). The amino acid sequence of the recombinant human IL-21 suitable for use in this invention is provided in Table 2 (SEQ ID NO:21).

When indicated as “effective antitumor dose,” “effective tumor-suppressing dose,” or “therapeutic dose,” the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease. Generally, it can be stated that the pharmaceutical composition described herein, comprising tumor-infiltrating lymphocytes (e.g., passaged TILs or genetically modified cytotoxic lymphocytes), can be administered at doses of ^10⁴ to ^10¹¹ cells/kg body weight (e.g., ^10⁵ to ^10⁶ , ^10⁵ to ^10¹⁰ , ^10⁵ to ^10¹¹ , ^10⁶ to ^10¹⁰ , ^{10⁶ to 10¹¹ , 10⁷} to ^10¹¹ , ^10⁷ to ^10¹⁰ , 10⁸ to ^10¹¹ , ^10⁸ to ^10¹⁰ , ^10⁹ to ^10¹¹ , or ^10⁹ to ^10¹⁰ cells/kg body weight), including all integer values within the range. TIL (in some cases, including genetically modified cytotoxic lymphocytes) compositions may also be administered at these doses multiple times. TILs (including genetically engineered TILs in some cases) can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). The optimal dosage and treatment regimen for a particular patient can be readily determined by medical professionals by monitoring the patient’s disease symptoms and adjusting the treatment accordingly.

The term "hematological malignancy" or related terms refer to cancers and tumors of the hematopoietic and lymphoid tissues of mammals (including, but not limited to, tissues of the blood, bone marrow, lymph nodes, and lymphatic system). Hematological malignancies are also known as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancy" refers to hematological malignancies affecting B cells.

The term "liquid tumor" refers to an abnormal clump of cells that is fluid in nature. Liquid tumor cancers include (but are not limited to) leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs derived from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs derived from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

As used herein, the term “microenvironment” can refer to the solid or hematologic tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanotransmitters that “promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, encourage treatment resistance, and provide a niche for the vigorous growth of dominant metastases,” as described by Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression of the microenvironment.

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (on days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m²/day for 5 days (on days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours to achieve physiological tolerance.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for components of the immune system (“cytokine pool”) prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as “immunosuppressive modulation”) in a patient prior to the introduction of the TIL of the present invention.

The term "effective amount" or "therapeutic effective amount" refers to an amount of compound or combination of compounds as described herein that is sufficient to achieve the intended application, including (but not limited to) the treatment of a disease. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo) or the subject being treated and the disease condition (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. The term also applies to doses that will induce a specific response in target cells (e.g., reduced platelet adhesion and/or cell migration). Specific doses will vary depending on: the specific compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

The terms “treatment”, “treating”, “treat”, etc., refer to achieving a desired pharmacological and/or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of partial or complete cure of a disease and/or adverse effects attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development or progression; and (c) alleviating the disease, i.e., causing disease remission and/or alleviating one or more disease symptoms. “Treatment” is also intended to cover the delivery of agents to provide a pharmacological effect, even in the absence of a disease or ailment. For example, “treatment” covers the delivery of compositions that can induce or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

The terms “non-myeloablative chemotherapy,” “non-myeloablative lymphocyte depletion,” “NMALD,” “NMA LD,” “NMA-LD,” and any variations thereof are used interchangeably to indicate a chemotherapy regimen designed to deplete a patient’s lymphoid immune cells while avoiding depletion of their myeloid immune cells. Typically, a patient receives one cycle of non-myeloablative chemotherapy before receiving tumor-infiltrating lymphocytes as described herein.

When used in conjunction with parts of a nucleic acid or protein, the term "heterologous" indicates that the nucleic acid or protein contains two or more subsequences that are not found in nature to be identical to each other. For example, nucleic acids are often produced in a recombinant manner, having two or more sequences from unrelated genes arranged to create new functional nucleic acid sequences, such as a promoter from one source and a coding region from another source or coding regions from different sources. Similarly, a heterologous protein indicates a protein that contains two or more subsequences that are not found in nature to be identical to each other (e.g., fusion proteins).

In the context of two or more nucleic acids or polypeptides, the terms "sequence identity," "percent identity," and "sequence percent identity" (or their synonyms, such as "99% identical") refer to two or more sequences or subsequences that are identical or have the same specific percentage of nucleotide or amino acid residues when compared and aligned (introducing gaps where necessary) to achieve maximum correspondence and without considering any conserved amino acid substitutions as part of the sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. Suitable programs for determining the percentage of sequence identity include, for example, the BLAST package available from the U.S. Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California, USA), or MegAlign (available from DNASTAR) are other publicly available software programs that can be used for sequence alignment. Those skilled in the art can determine the appropriate parameters for optimal alignment using specific alignment software. In some embodiments, the default parameters of the alignment software are used.

As used herein, the term "variant" covers (but is not limited to) antibodies or fusion proteins that contain an amino acid sequence different from that of a reference antibody, differing in that one or more substitutions, deletions, and/or additions are present within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conserved substitutions in its amino acid sequence compared to that of the reference antibody. Conserved substitutions may involve, for example, substitutions of similar charged or uncharged amino acids. Variants retain the ability to bind specifically to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

In this document, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are cells obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any population of expanded or proliferated TIL cells as discussed herein, including (but not limited to) the main TILs and expanded TILs (“REP TILs”) as discussed herein, as well as “reREP TILs”. reREP TILs may include, for example, a second expanded TIL or a second additional expanded TIL (e.g., the TILs described in step D of Figure 8, including TILs referred to as reREP TILs).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to be reintroduced into patients after infiltrating solid tumors. TILs can be further characterized by potency; for example, if interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, then TILs can be considered potent. TILs can be considered potent if, for example, interferon (IFNγ) releases greater than approximately 50 pg/mL, greater than approximately 100 pg/mL, greater than approximately 150 pg/mL, or greater than approximately 200 pg/mL, greater than approximately 300 pg/mL, greater than approximately 400 pg/mL, greater than approximately 500 pg/mL, greater than approximately 600 pg/mL, greater than approximately 700 pg/mL, greater than approximately 800 pg/mL, greater than approximately 900 pg/mL, or greater than approximately 1000 pg/mL.

The term "deoxyribonucleic acid" encompasses both natural and synthetic, unmodified and modified deoxyribonucleic acids. Modifications include alterations to the linkages between deoxyribonucleic acids in the sugar moiety, base moiety, and/or oligonucleotides.

The term "RNA" is defined as a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" is defined as a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, RNA produced in a recombinant manner, and altered RNA that differs from naturally occurring RNA through the addition, deletion, substitution, and/or modification of one or more nucleotides. The nucleotides in the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, as well as inert components. The use of such pharmaceutically acceptable carriers or excipients for the active pharmaceutical ingredient is well known in the art. Unless any conventionally pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is covered. Additional active pharmaceutical ingredients, such as those of other pharmaceuticals, may also be incorporated into the described compositions and methods.

The terms “about” and “approximately” mean a range of values that are statistically significant. This range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The permissible differences covered by the terms “about” or “approximately” depend on the specific system under study and are readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not precise and do not need to be precise, but may be approximate and/or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. Generally, whether so explicitly stated or not, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are “about” or “approximately”. It should be noted that embodiments with very different sizes, shapes, and dimensions may employ the described arrangement.

When used in the appended claims in both original and modified forms, the transitional terms “comprising,” “consisting essentially of,” and “consisting of” define the scope of the invention with respect to which additional, unrecited elements or steps (if present) are excluded from the scope of the claims. The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case excludes impurities generally associated with the specified materials. The term “consisting essentially of” limits the scope of the invention to the specified elements, steps, or materials and those elements, steps, or materials that do not substantially affect the basis and novel features of the claimed invention. In alternative embodiments, all compositions, methods, and kits for practicing the invention described herein may be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

The term “antibody” and its plural form “antibodies” refer to a complete immunoglobulin and any antigen-binding fragment (“antigen-binding moiety”) or its single chain. “Antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or its antigen-binding moiety. Each heavy chain includes a heavy chain variable region (abbreviated as _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated as _VL ) and a light chain constant region. The light chain constant region comprises one domain, _CL . The _VH and _VL regions of an antibody can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), and these may be interspersed with more conserved regions called frame regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more epitopes. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen" refers to a substance that induces an immune response. In some embodiments, if presented via a major histocompatibility complex (MHC) molecule, the antigen is a molecule capable of being bound by an antibody or TCR. As used herein, the term "antigen" also encompasses T-cell epitopes. Antigens are additionally capable of being recognized by the immune system. In some embodiments, the antigen is capable of inducing a humoral or cellular immune response that results in the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require the antigen to contain or be linked to a Th-cell epitope. The antigen may also have one or more epitopes (e.g., B epitopes and T epitopes). In some embodiments, the antigen is preferably to react generally with its corresponding antibody or TCR in a highly specific and selective manner, and not with a variety of other antibodies or TCRs that can be induced by other antigens.

The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to formulations of antibody molecules that are single molecular compositions. Monoclonal antibody compositions exhibit single binding specificity and affinity against a specific epitope. Monoclonal antibodies specific to certain receptors can be prepared using knowledge and techniques in the following methods: injecting a suitable antigen into a test subject and then isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. DNA encoding monoclonal antibodies can be isolated and sequenced using routine procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not normally produce immunoglobulins (e.g., *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to obtain monoclonal antibody synthesis in recombinant host cells. The recombinant production of antibodies will be described in more detail below.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Antigen-binding function of antibodies has been demonstrated to be performed by fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of _VL , _VH , _CL , and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; (iii) Fd fragments consisting of _VH and CH1 domains; (iv) Fv fragments consisting of _VL and _VH domains of a single arm of an antibody; (v) domain antibody (dAb) fragments (Ward et al., Nature, 1989, 341, 544-546), which may consist of a _VH or a _VL domain; and (vi) segregated complementarity-determining regions (CDRs). Furthermore, although the two domains ( _VL and _VH ) of the Fv fragment are encoded by independent genes, they can be joined using recombination methods via a synthetic adapter that enables the creation of a single protein chain (referred to as a single-chain Fv (scFv)) in which the _VL and _VH regions pair to form a monovalent molecule; see, for example, Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are targeted at the utility screening fragment in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises both the _VH and _VL portions. A scFv molecule is represented as _VL - _LVH when the _VL domain is the N-terminal portion of the scFv molecule, or as _VH - _LVL when the _VH domain is the N-terminal portion of the scFv molecule. Methods for preparing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB, Vol. 9: 73-80 (1995); and REBird and BWWalker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9: 132-137 (1991), the disclosures of which are incorporated herein by reference.

As used herein, the term "human antibody" is intended to include antibodies that have variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, said constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human framework sequence.

The term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity with a variable region, wherein both the framework and CDR regions of the variable region are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is generated from a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with immortalized cells, having a genome containing both human heavy-chain and light-chain transgenes.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas prepared therefrom (described further below) that are transgenic for the human immunoglobulin gene; (b) antibodies isolated from host cells transformed to express human antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may undergo in vitro mutation induction (or in vivo somatic mutation induction when using transgenic animals with human Ig sequences), and therefore the amino acid sequences of the _VH and _VL regions of the recombinant antibody, although derived from and associated with human germline _VH and _VL sequences, may not be naturally present in the human antibody germline in vivo.

As used in this article, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

The term "human antibody derivative" refers to any modified form of a human antibody, including conjugates of antibodies with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which may be conjugated to the antibody described herein using methods available in the art.

The terms "humanized antibody" and "humanized" refer to antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence. Further frame region modifications can be made to the human frame sequence. Humanized forms of non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. To a large extent, humanized antibodies are human immunoglobulins (receptor antibodies) where residues from the hypervariable region of the receptor are replaced by residues from 15 hypervariable regions of a non-human species (donor antibody) possessing the desired specificity, affinity, and ability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in receptor or donor antibodies. These modifications are performed to further optimize antibody performance. Generally, humanized antibodies will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially all FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein may also be modified to use any Fc variant known to provide effector function and/or FcR binding improvements (e.g., reductions). Fc variants may include any of the amino acid substitutions disclosed below: International Patent Application Publications No. WO 1988/07089A1, WO 1996/14339A1, WO 1998/05787A1, WO 1998/23289A1, WO 1999/51642A1, WO 99/58572A1, WO 2000/09560A2, WO 2000/32767A1, WO 20 No. 00/42072A2, No. WO 2002/44215A2, No. WO 2002/060919A2, No. WO 2003/074569 A2, No. WO 2004/016750 A2, No. WO 2004/029207 A2, No. WO 2004/035752 A2, No. WO 2004/063351A2, No. WO 2004/074455 A2, No. WO 2004/09924 9 A2, WO 2005/040217 A2, WO 2005/070963 A1, WO 2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 A1, WO 2006/047350 A2 and WO 2006/085967 A2; and U.S. Patent No. 5,648,260; Nos. 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the contents of which are incorporated herein by reference.

The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

A “bifunctional antibody” is a small antibody fragment having two antigen-binding sites. The fragment contains a heavy chain variable domain (V_{_H} ), which is linked to a light chain variable domain (V_L ) in the same polypeptide chain (V_{_H}-V_{_L} or V_{_L}-V_{_H}). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, thus creating two antigen-binding sites. Bifunctional antibodies are described in more detail, for example, in European Patent No. 404,097, International Patent Publication No. WO 93/11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

The term "glycosylation" refers to a modified derivative of an antibody. Deglycosylated antibodies are not glycosylated. Glycosylation can be altered to, for example, increase the antibody's affinity for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at said sites. Deglycosylation can increase the antibody's affinity for an antigen, as described in U.S. Patents 5,714,350 and 6,350,861. Alternatively or additionally, antibodies with altered glycosylation types can be produced, such as low-fucosylated antibodies with reduced fucosylation residues or antibodies with increased fractional GlcNac structure. Such altered glycosylation patterns have been shown to enhance antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention to produce glycosylated-modified antibodies. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)fucosyltransferase), so that antibodies expressed in Ms704, Ms705, and Ms709 cell lines do not have fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/- cell lines were formed by breaking the FUT8 gene in targeted CHO/DG44 cells using two alternative vectors (see, for example, U.S. Patent Publication No. 2004/0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation by reducing or eliminating α1,6 bond-related enzymes. The patent also describes cell lines with low or no enzymatic activity for adding fucose to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). International Patent Publication WO 03/035835 describes a variant CHO cell line, namely Lec 13 cells, which has a reduced ability to link fucose to Asn(297)-linked carbohydrates and also induces hypofucosylation of antibodies expressed in said host cells (see also Shields et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication WO 99/54342 describes cells engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine). Cell lines containing GnTIII transferase III (GnTIII) can be used to enhance the antibody's ADCC activity by causing antibodies expressed in engineered cell lines to exhibit an increased bipartite GlcNac structure (see also Umana et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidases can be used to cleave the fucosylate residues of the antibody. For example, α-L-fucosidase removes fucosylate residues from the antibody, as described in Tarentino et al., Biochem. 1975, 14, 5516-5523.

“Polyglycolization” refers to the modification of an antibody or fragment thereof, typically with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that link one or more PEG groups to the antibody or antibody fragment. For example, PEGylation can increase the biological (e.g., serum) half-life of an antibody. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to cover any form of PEG that has been used to derive other proteins, such as mono( _C1 - _C10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-cis-butenediamide. The antibody to be PEGylated is a deglycosylated antibody. Polyethylene glycolation methods are known in the art and applicable to antibodies used in this invention, such as those described, for example, in European Patents EP0154316 and EP0401384 and U.S. Patent No. 5,824,778, the disclosures of which are each incorporated herein by reference.

The term "biosimilar" refers to a biological product (including monoclonal antibodies or proteins) that meets the following criteria: it is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, and there are no clinically significant differences between the biological product and the reference product in terms of safety, purity, and efficacy. Furthermore, a biosimilar or "biosimilar" drug is a biological drug similar to another biological drug authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. A biological product or biological drug is a drug derived from or produced from a biological source (e.g., bacteria or yeast). It may consist of relatively small molecules (e.g., human insulin or erythropoietin) or complex molecules (e.g., monoclonal antibodies). For example, if the reference IL-2 protein is proleukin, then the protein of the reference proleukin approved by a drug regulatory agency is "biosimilar to proleukin" or "a biosimilar of proleukin." In Europe, a biosimilar or “biosimilar” medicine is a biological medicine similar to another biological medicine that has been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for the use of biosimilars in Europe is Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended, and therefore in Europe, biosimilars may be authorized, approved for authorization, or submitted as the subject of authorization pursuant to Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC. The original biological medicine product authorized in Europe may be referred to as a “reference medicine.” The CHMP guidance on biosimilar products outlines some of the requirements for a product to be considered a biosimilar. In addition, product-specific guidance, including guidance relating to monoclonal antibody biosimilars, is provided by the EMA on a product-by-product basis and published on its website. Biosimilars as described herein may be similar to a reference medicine in terms of quality characteristics, biological activity, mechanism of action, safety profile, and/or efficacy. Furthermore, biosimilars may be used or intended for the treatment of the same disease as the reference medicine. Therefore, biosimilars as described herein can be considered to have similar or very similar quality characteristics to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar biological activity to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar safety profile to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar efficacy to the reference drug. As described herein, in Europe, biosimilars have been compared with reference drugs authorized by the EMA. However, in some cases, in certain studies, biosimilars may be compared with biological products authorized outside the European Economic Area (non-EEA authorized "comparatives"). Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to biological products that have been or may be compared with non-EEA authorized comparatives. Some biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding moieties), and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications (including, for example, amino acid deletions, additions, and/or substitutions) in their amino acid structure that do not significantly affect peptide function. Biosimilars may contain an amino acid sequence having 97% or higher, such as 97%, 98%, 99%, or 100%, sequence identity with the amino acid sequence of a reference drug. Biosimilars may contain one or more post-translational modifications that differ from those of the reference drug, such as (but not limited to) glycosylation, oxidation, deamidation, and/or truncation, provided that such differences do not alter the safety and/or efficacy of the drug. Biosimilars may have the same or different glycosylation patterns as the reference drug. In particular, but not exclusively, biosimilars may have different glycosylation patterns if such differences resolve or are intended to resolve safety concerns associated with the reference drug. Additionally, biosimilars may differ from the reference drug in, for example, their potency, pharmaceutical form, formulation, excipients, and/or delivery method, provided that this does not impair the safety and efficacy of the drug. Compared to a reference product, biosimilars may contain differences in, for example, pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles, but are still considered sufficiently similar to the reference product and thus eligible for or suitable for authorization. In some cases, biosimilars exhibit different binding characteristics compared to the reference product, where the regulatory agency (e.g., the EMA) does not consider these different binding characteristics as an obstacle to authorization of the biosimilar product. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions.

II. Gene Editing Process

A. Overview: TIL amplification + gene editing

Embodiments of the present invention relate to methods for expanding a TIL population, said methods comprising one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, “gene-editing” and “genome editing” refer to a gene modification in which DNA is permanently modified in the genome of a cell, such as by inserting, deleting, modifying, or replacing DNA within the cell's genome. In some embodiments, gene editing causes silencing (sometimes referred to as gene knockout) or suppression/reduction (sometimes referred to as gene knockdown) of DNA sequence expression. According to embodiments of the present invention, gene editing technology is used to enhance the effectiveness of a therapeutic TIL population.

Methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the methods further include gene editing of at least a portion of the TILs. According to other embodiments, methods for expanding TILs into a therapeutic TIL population can be carried out according to any embodiment of the methods described in WO 2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, which are incorporated herein by reference in their entirety, wherein the methods further include gene editing of at least a portion of the TILs. Therefore, one embodiment of the invention provides a therapeutic TIL population expanded according to any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, for example, at least a portion of the therapeutic TIL population transferred to an infusion bag has been permanently gene-edited.

Gene editing during B.TIL amplification

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the method includes the step of culturing or initially expanding a first TIL population, comprising culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some embodiments, the method includes the steps of culturing or rapidly expanding the third TIL population by culturing the third TIL population in a second cell culture medium for a first period of about 1-7 days, splitting the culture into multiple subcultures at the end of the first period, culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 3-7 days, and merging the multiple subcultures at the end of the second period to provide an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the activation of the second TIL population takes approximately 7 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during, or before, any of steps (a)-(e), (a)-(f), or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e), (a)-(f), or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A third population of TILs was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some embodiments, the step of culturing the third TIL population takes approximately 7 days. In some embodiments, the step of culturing the third TIL population takes approximately 8 days. In some embodiments, the step of culturing the third TIL population takes approximately 9 days. In some embodiments, the step of culturing the third TIL population takes approximately 10 days. In some embodiments, the step of culturing the third TIL population takes approximately 11 days. In some embodiments, the step of culturing the third TIL population takes approximately 12 days. In some embodiments, the step of culturing the third TIL population takes approximately 13 days. In some embodiments, the step of culturing the third TIL population takes approximately 14 days. In some embodiments, the step of culturing the third TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(d) or (a)-(e) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(d) or (a)-(e), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some implementation schemes, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementation schemes, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are then placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 1-7 days. In some implementations, the step of culturing the third TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the third TIL population takes approximately 1 day. In some implementations, the step of culturing the third TIL population takes approximately 2 days. In some implementations, the step of culturing the third TIL population takes approximately 3 days. In some implementations, the step of culturing the third TIL population takes approximately 4 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some implementations, the process of culturing a third TIL population takes about 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed over a period of approximately 22 days. In some embodiments, the steps of the method are completed over a period of approximately 23 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some implementations, the steps of the method are completed over a period of approximately 22 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1 day. In some implementations, the step of culturing the fourth TIL population takes approximately 2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 3 days. In some implementations, the step of culturing the fourth TIL population takes approximately 4 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, initial amplification takes approximately 3-9 days. In other implementations, initial amplification takes approximately 1-9 days, 2-9 days, 3-9 days, approximately 4-9 days, approximately 5-9 days, approximately 6-9 days, approximately 7-9 days, approximately 8-9 days, approximately 1-8 days, approximately 2-8 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, initial amplification takes approximately 1 day. In some embodiments, initial amplification takes approximately 2 days. In some embodiments, initial amplification takes approximately 3 days. In some embodiments, initial amplification takes approximately 4 days. In some embodiments, initial amplification takes approximately 5 days. In some embodiments, initial amplification takes approximately 6 days. In some embodiments, initial amplification takes approximately 7 days. In some embodiments, initial amplification takes approximately 8 days. In some embodiments, initial amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the rapid second amplification takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error or incomplete sentence.) The durations of rapid secondary amplification are approximately 11-13 days, 12-13 days, 5-12 days, 6-12 days, 7-12 days, 8-12 days, 9-12 days, 10-12 days, 11-12 days, 5-11 days, 6-11 days, 7-11 days, 8-11 days, 9-11 days, 10-11 days, 5-10 days, 6-10 days, 7-10 days, 8-10 days, 9-10 days, 5-9 days, 6-9 days, 7-9 days, 8-9 days, 5-8 days, 6-8 days, 7-8 days, 5-7 days, 6-7 days, and 5-6 days. In some implementations, rapid secondary amplification takes approximately 5 days. In some implementations, rapid secondary amplification takes approximately 6 days. In some implementations, rapid secondary amplification takes approximately 7 days. In some embodiments, rapid second amplification takes approximately 8 days. In some embodiments, rapid second amplification takes approximately 9 days. In some embodiments, rapid second amplification takes approximately 10 days. In some embodiments, rapid second amplification takes approximately 11 days. In some embodiments, rapid second amplification takes approximately 12 days. In some embodiments, rapid second amplification takes approximately 13 days. In some embodiments, rapid second amplification takes approximately 14 days. In some embodiments, rapid second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digest the tumor tissue sample or tumor fragment in an enzyme medium to produce tumor digest;

(c) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some implementations, the first amplification takes approximately 3-9 days. In some implementations, the first amplification takes approximately 3-9 days, approximately 3-8 days, approximately 3-7 days, approximately 3-6 days, approximately 3-5 days, approximately 3-4 days, approximately 4-9 days, approximately 4-8 days, approximately 5-9 days, approximately 5-8 days, approximately 6-9 days, approximately 6-8 days, approximately 7-9 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the first amplification takes approximately 3 days. In some implementations, the first amplification takes approximately 4 days. In some implementations, the first amplification takes approximately 5 days. In some implementations, the first amplification takes approximately 6 days. In some implementations, the first amplification takes approximately 7 days. In some implementations, the first amplification takes approximately 8 days. In some implementations, the first amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes approximately 5-15 days. In some implementations, the second amplification takes approximately 5-15 days, approximately 6-15 days, approximately 7-15 days, approximately 8-15 days, approximately 9-15 days, approximately 10-15 days, approximately 11-15 days, approximately 12-15 days, approximately 13-15 days, approximately 14-15 days, approximately 5-14 days, approximately 6-14 days, approximately 7-14 days, approximately 8-14 days, approximately 9-14 days, approximately 10-14 days, approximately 11-14 days, approximately 12-14 days, approximately 13-14 days, approximately 5-13 days, approximately 6-13 days, approximately 7-13 days, approximately 8-13 days, approximately 9-13 days, approximately 10-13 days. The durations of amplification are as follows: approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the second amplification takes approximately 5 days. In some implementations, the second amplification takes approximately 6 days. In some implementations, the second amplification takes approximately 7 days. In some implementations, the second amplification takes approximately 8 days. In some embodiments, the second amplification takes approximately 9 days. In some embodiments, the second amplification takes approximately 10 days. In some embodiments, the second amplification takes approximately 11 days. In some embodiments, the second amplification takes approximately 12 days. In some embodiments, the second amplification takes approximately 13 days. In some embodiments, the second amplification takes approximately 14 days. In some embodiments, the second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days. In some embodiments, the steps of the method are completed within a period of approximately 32 days.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

In some implementations, gene editing is performed while the TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementations, TILs collected from the culture medium are gene-edited, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, the step of gene editing at least a portion of the second or third TIL population includes a sterile electroporation step on the second or third TIL population.

In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some embodiments, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step further includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 22 hours. According to some implementations, the quiescent step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the antigen-presenting cells (APCs) are PBMCs. According to some embodiments, the PBMCs are irradiated. According to some embodiments, the PBMCs are allogeneic. According to some embodiments, the PBMCs are both irradiated and allogeneic. According to some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some implementations, the tumor tissue is derived from an autopsied tumor.

In some implementations, the dissected tumor is less than 8 hours old.

In some embodiments, the tumor tissue is selected from the group consisting of: melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple-negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and HPV-positive tumor tissue.

In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 2 mm.

In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 1.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5.5 mm and a longest side length of about 6 mm.

In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm or about 6 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 6 mm.

In some embodiments, the present invention provides a therapeutic population of tumor-infiltrating lymphocyte (TIL) products generated by methods as described herein.

In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising administering to the patient an effective amount of a therapeutic TIL population generated by the method described herein. In some embodiments, the cancer is selected from the group consisting of: glioblastoma (GBM), gastrointestinal cancer, melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, and mantle cell lymphoma. In some implementations, the cancer is selected from the group consisting of: cutaneous melanoma, ocular melanoma, uveal melanoma, conjunctival malignant melanoma, metastatic melanoma, pleomorphic xanthoastrocytoma, dysembryonic neuroepithelial tumor, ganglioglioma and pilocytic astrocytoma, endometrioid adenocarcinoma with significant myxoid differentiation (ECMD), papillary thyroid carcinoma, serous low-grade or borderline ovarian cancer, hairy cell leukemia and Langerhans cell histiocytosis.

In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 2000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 2000 IU/mL.

In some embodiments, in the second amplification step, the IL-2 is present at an initial concentration between 1000 IU/mL and 6000 IU/mL, and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some embodiments, the first amplification is performed using a ventilated container. In some embodiments, the second amplification is performed using a ventilated container.

In some embodiments, the first cell culture medium further comprises cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and/or the third culture medium further comprise cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the method further includes the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated the day after administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated on the same day as administration of the TIL or PBL product. In some embodiments, the IL-2 regimen comprises adelleukin, nevoleukin, or a bioanalyte or variant thereof.

In some embodiments, the therapeutically effective amount of TIL product comprises about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs.

In some implementations, the number of the second TIL population is at least 50 times that of the first TIL population.

C.PD-1

One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 superfamily of T cell regulators. Its ligands, PD-L1 and PD-L2, are expressed on various tumor cells, including melanoma. The interaction between PD-1 and PD-L1 can suppress T cell effector function, cause T cell exhaustion under chronic stimulation, and induce T cell apoptosis in the tumor microenvironment. PD-1 may also play a role in tumor-specific evasion of immune surveillance.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of PD-1 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the method includes gene editing of at least a portion of the TILs by silencing or inhibiting the expression of PD-1. As described in more detail below, the gene editing process may involve the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., PD-1). For example, the TALEN method can be used to silence or reduce the expression of PD-1 in TILs.

D.CTLA-4

CTLA-4 expression is induced upon T cell activation on activated T cells and competitively binds to antigen-presenting cell activation antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 can induce T cell suppression and is used to maintain the homeostasis of the immune response. However, inhibiting the interaction of CTLA-4 with CD80 or CD86 prolongs T cell activation and thus increases the level of the immune response against cancer antigens.

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of CTLA-4 in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and CTLA-4 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CTLA-4. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CTLA-4). For example, the CRISPR method, the TALE method, or the zinc finger method can be used to silence or inhibit the expression of CTLA-4 in TILs. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and CTLA-4 in TILs.

E.LAG-3

Following conjugation at the major histocompatibility complex (MHC) class II, lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells. Although the mechanism is not fully understood, regulation of this gene can induce negative regulation of T cell function, preventing tissue damage and autoimmunity. Therefore, LAG-3 blockade may improve antitumor responses. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of LAG-3 in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and LAG-3 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of LAG-3. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., LAG-3). According to specific embodiments, the expression of LAG-3 in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and LAG-3 in TILs can be silenced or reduced using the TALEN method.

F.Cish

Cish, a member of the cytokine signaling inhibitory factor (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits its functional affinity for tumors. Deletion of the Cish gene in CD8+ T cells enhances its amplification, functional affinity, and cytokine pluripotency, leading to significant and durable regression of existing tumors. See, for example, Palmer et al., Journal of Experimental Medicine, 212(12):2095 (2015).

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of Cish in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and Cish in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of Cish. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., Cish). For example, the expression of Cish in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and Cish in TILs can be silenced or reduced using the TALEN method.

G.CBL-B

CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and a negative regulator of T cell activation. (Bachmaier et al., Nature, 2000, 403, 211-216; Wallner et al., Clin. Dev. Immunol. 2012, 692-639.)

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of CBL-B in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and CBL-B in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., process 2A or the method shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CBL-B. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CBL-B). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, CBL-B is silenced using TALEN gene knockout. In some embodiments, CBL-B is silenced using a TALE-KRAB transcription inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and CBL-B in TIL.

H.TIGIT

TIGIT is a cell surface protein expressed on regulatory, memory, and activated T cells. TIGIT belongs to the poliovirus receptor (PVR) family of immunoglobulins and inhibits T cell activation. (Yu et al., Nat Immunol., 2009, 10(1):48-57).

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of TIGIT in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and TIGIT in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of TIGIT. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., TIGIT). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, TIGIT can be silenced using TALEN gene knockout. In some embodiments, TIGIT can be silenced using a TALE-KRAB transcriptional inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and TIGIT in TIL.

III. Gene Editing Methods

As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) genetically modified via gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing performed via nucleotide insertion (RNA or DNA) into a TIL population to suppress the expression of one or more proteins. Embodiments of the present invention also provide a method for expanding TILs into a therapeutic population, wherein the method includes gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, and these gene editing techniques are suitable for use according to the present invention.

In some embodiments, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes a retroviral transduction step. In one embodiment, the method of genetically modifying a TIL population includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, a method for genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which a transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long-term expression of the transposase does not occur in transgenic cells, for example, a transposase provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems include salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In one embodiment, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing or repressing (e.g., silencing) one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In another embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating TILs with a pulsed electric field to induce pore formation in the TILs, including the step of applying a series of at least three DC electrical pulses to the TILs, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time and such that the survival rate of the TILs is maintained. In one embodiment, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes the step of transfection using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

According to one embodiment, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems, such as Cas9, target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA bases and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, a method for amplifying TILs into a therapeutic population may be performed according to any embodiment of the method described herein (e.g., procedure 2A) or as described in WO2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, wherein said method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by comparing gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

1. TALE method

A method for amplifying TILs into a therapeutic population can be made according to any embodiment of the method described herein or as described in WO 2018/081473 A1, WO 2018/129332A1, or WO 2018/182817 A1, wherein the method further includes gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the use of the TALE method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the TALE method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for “transcription activator-like effector” protein, which includes TALEN (“transcription activator-like effector nuclease”). The method of gene editing using the TALE system is also referred to herein as the TALE method. TALE is a naturally occurring protein from the plant pathogenic bacterium *Xanthomonas* and contains a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize continuous DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by 14–20 base-pair spacers, bring FokI monomers closer together for dimerization and to generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and T NFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA , IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

Non-limiting examples of TALE-nucleases targeting the PD-1 gene are provided in the table below. In these examples, the targeted genomic sequence contains two 17-base-pair (bp) long sequences (referred to as half-targets, shown in uppercase letters) separated by a 15-bp spacer. Each half-target is recognized by a repetitive sequence of the half-TALE-nuclease listed in the table. Thus, according to a particular embodiment, the TALE-nuclease according to the invention recognizes and cleaves target sequences selected from the group consisting of: SEQ ID NO:127 and SEQ ID NO:128. TALEN sequences and gene editing methods are also described in Gautron et al., Molecular Therapy: Nucleic Acids, December 2017, Vol. 9: 312-321, which is incorporated herein by reference.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

2. Cas-CLOVER method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs using a Cas-CLOVER method. According to a particular embodiment, the use of the Cas-CLOVER method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the Cas-CLOVER method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Cas-CLOVER is a dimerizing, high-fidelity site-specific nuclease (SSN) composed of a fusion of catalytically dead SpCas9 (dCas9) and a nuclease domain from a Clostridium Clo051 type IIs restriction endonuclease (Madison et al., “Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched alloeneic CAR-T cells”, Molecular Therapy-Nucleic Acids, 2022). This produces a nuclease whose activity is determined by the dimerization of the Clo051 nuclease domain, which recognizes two adjacent 20-nt target sequences via RNA guidance. Unlike paired nicking enzyme approaches, for example, when using the Cas9-D10A mutant, monomeric Cas-CLOVER does not introduce nicks or DSBs. Cas-CLOVER has been shown to have low off-target nuclease activity.

Exemplary Cas-CLOVER systems include those described in WO2019/126578, the contents of which are incorporated herein by reference in their entirety. In embodiments, the Cas-CLOVER system comprises a fusion protein containing a DNA-localizing component and an effector molecule, substantially consisting of or consisting of therein.

DNA positioning components

In this embodiment, the DNA targeting component is capable of binding to a specific DNA sequence. In this embodiment, the DNA targeting component is selected from, for example, DNA-binding oligonucleotides, DNA-binding proteins, DNA-binding protein complexes, and combinations thereof. Other suitable DNA-binding components will be recognized by those skilled in the art.

In one embodiment, the DNA targeting component comprises oligonucleotides targeting one or more specific loci in the genome. The oligonucleotides may be selected from DNA, RNA, DNA/RNA hybrids, and combinations thereof.

In some embodiments, the DNA positioning component comprises a nucleotide-binding protein or protein complex that binds an oligonucleotide upon binding to the target DNA. The protein or protein complex may be able to recognize features selected from RNA-DNA heteroduplexes, R-loops, or combinations thereof. In some embodiments, the DNA positioning component comprises a protein or protein complex capable of recognizing an R-loop selected from Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, or combinations thereof. In some embodiments, the DNA positioning component comprises an engineered protein capable of binding to the target DNA. In some embodiments, the DNA positioning component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, transcription activator-like (TAL) arrays, and combinations thereof. In some embodiments, the DNA positioning component comprises a protein containing a naturally occurring DNA-binding domain. In some embodiments, the DNA positioning component comprises a bZIP domain, a helix-loop-helix, a helix-turn-helix, an HMG-box, a leucine zipper, a zinc finger, or combinations thereof. In some embodiments, the DNA positioning component comprises an oligonucleotide targeting a specific locus in the genome. Exemplary oligonucleotides include, but are not limited to, DNA, RNA, DNA/RNA hybrids, and any combination thereof. In some embodiments, the DNA targeting component comprises a protein or protein complex capable of recognizing features selected from RNA-DNA heteroduplexes, R-loops, and any combinations thereof. Exemplary proteins or protein complexes capable of recognizing R-loops include, but are not limited to, Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, and any combinations thereof. In some embodiments, the protein or protein complex capable of recognizing R-loops comprises Cas9. In some embodiments, the DNA targeting component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, TAL arrays, and any combinations thereof. In some embodiments, the DNA targeting component comprises an oligonucleotide targeting a specific location in the genome and a protein capable of binding to a target DNA sequence.

In one embodiment, the DNA positioning component comprises at least one guide RNA (gRNA), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises two gRNAs, substantially consisting of or consisting of them, wherein the first gRNA specifically binds to the first strand of the double-stranded DNA target sequence, and the second gRNA specifically binds to the second strand of the double-stranded DNA target sequence. Alternatively, in one embodiment, the DNA positioning component comprises a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Ralstonia*, substantially consisting of or consisting of it.

Effect molecules

In the implementation scheme, the effector molecule is capable of producing a predetermined effect at a specific locus in the genome. Exemplary effector molecules include, but are not limited to, transcription factors (activators or repressors), chromatin remodeling factors, nucleases, exonucleases, endonucleases, transposases, methyltransferases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, helicases, fluorophores, or any combination thereof.

In some embodiments, the effector molecule comprises a transposase. In some embodiments, the effector molecule comprises PB transposase (PBase). In some embodiments, the effector molecule comprises a nuclease. Non-limiting examples of nucleases include restriction endonucleases, homing endonucleases, S1 nucleases, mung bean nucleases, pancreatic DNase I, micrococcal nucleases, yeast HO endonucleases, or any combination thereof. In some embodiments, the effector molecule comprises a restriction endonuclease. In some embodiments, the effector molecule comprises a type IIS restriction endonuclease. In some embodiments, the effector molecule comprises a nuclease. Non-restrictive examples of endonucleases include AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, and Clo051. In the implementation scheme, the effector molecule includes BmrI, BfiI, or Clo051.

In embodiments, the effector molecule comprises a homodimer or heterodimer, substantially composed of, or composed of. In embodiments, the effector molecule comprises a nuclease, optionally an endonuclease, substantially composed of, or composed of. In embodiments, the effector molecule, including those comprising a homodimer or heterodimer, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially composed of, or composed of. In embodiments, Cas9 is catalytically inactive or “inactivated” Cas9 (dCas9 (SEQ ID NO: 302 and 303 of WO2019/126578)). In embodiments, Cas9 is the catalytically inactive or “inactivated” nuclease domain of Cas9. In embodiments, dCas9 is encoded by a shorter sequence derived from full-length, catalytically inactivated Cas9, referred herein as “small” dCas9 or dSaCas9 (SEQ ID NO: 23 of WO2019/126578).

In embodiments of the fusion protein, the effector molecule comprises, is substantially composed of, or is composed of, one or more type II nucleases, homodimers or heterodimers of type II nucleases. In the implementation scheme, the type II nuclease includes one or more of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof that forms a heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises a catalytically inactive form of Cas9 (e.g., dCas9 or dSaCas9) or a fragment thereof that forms a heterodimer with Clo051, and is substantially composed of or constitutes thereof. An exemplary Clo051 nuclease domain may comprise the following amino acid sequence, and is substantially composed of or constitutes thereof:

EGIKSNISLLKDELRGQISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE

KIRSGEMTIEELERAMFNNSEFILKY(SEQ ID NO:238).

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces*, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, and is substantially composed of or constitutes thereof.

connect

In some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule, substantially consisting of or consisting of the latter. In some embodiments, nucleic acid sequences encoding one or more components of the fusion protein can be operatively linked, for example, to an expression vector. In some embodiments, the fusion protein is a chimeric protein. In some embodiments, the fusion protein is encoded by one or more recombinant nucleic acid sequences. In some embodiments, the fusion protein further includes a linker region to operatively link the two components of the fusion protein. For example, in some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule operatively linked by a linker region, substantially consisting of or consisting of the latter. In some embodiments, the DNA positioning component, linker region, and effector molecule can be encoded by one or more nucleic acid sequences inserted into an expression cassette and/or expression vector, such that translation of the nucleic acid sequences produces the fusion protein. In some embodiments, the fusion protein can include a non-covalent link between the DNA positioning component and the effector molecule. The non-covalent link can comprise an antibody, antibody fragment, antibody mimic, or scaffold protein.

Fusion protein

In one embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated Cas9 (dCas9) or an inactivated nuclease domain, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated small Cas9 (dSaCas9), substantially consisting of or consisting of it. In yet another embodiment, the effector molecule comprises Cas9, dCas9, dSaCas9, or a nuclease domain thereof and a second endonuclease, substantially consisting of or consisting of it. The second endonuclease may include, or be substantially composed of, type IIS endonucleases, including but not limited to, AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, and BmgBI. One or more of BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI, or Clo051.

In embodiments of the fusion protein, the DNA localization component comprises, is substantially composed of, or is composed of, a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), and the effector molecule comprises, substantially composed of, or is composed of, a nuclease. In embodiments of the fusion protein of this disclosure, the DNA localization component comprises, substantially composed of, or is composed of, a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rhodotorula*, and the effector molecule comprises, substantially composed of, or is composed of, an IIS-type nuclease, including but not limited to AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My One or more of I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In some embodiments, the exemplary dCas9-Clo051 fusion protein may comprise, substantially consist of, or consist of the amino acid sequence of SEQ ID NO:305 or 307 of WO2019/126578 or the nucleic acid sequence of SEQ ID NO:306 or 308 of WO2019/126578.

Construct

In the embodiments, the nuclease domain comprises dCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises dSaCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Xanthomonas spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Rocheborax spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the fusion protein comprises dCas9-Clo051, dSaCas9-Clo051, Xanthomonas spp.-TALE-Clo051, or Rocheborax spp.-TALE-Clo051. In the embodiments, the vector encoding the fusion protein comprises Csy4-T2A-Clo051-G4S linker-dCas9 (Streptococcus pyogenes) or pRT1-Clo051-dCas9 dual NLS.

According to some implementations, the Cas-CLOVER system comprises a fusion protein containing a DNA localization component and an effector molecule, wherein the DNA localization component hybridizes to a target sequence of a DNA molecule in a TIL, wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule, and the effector molecule cleaves the DNA molecule, thereby altering the expression of at least one immune checkpoint molecule.

According to a specific implementation, the Cas-CLOVER method involves silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas-CLOVER system (e.g., dCas9-Clo051, dSaCas9-Clo051, Xanthomonas-TALE-Clo051, or Rochetomyces-TALE-Clo051 fusion protein) that is specific to the target DNA sequence of immune checkpoint genes. The fusion protein can be delivered as DNA, mRNA, or protein. After the genome comes into contact with the Cas-CLOVER system, one or more strands of the target double-stranded DNA can be cleaved. If cleavage occurs in the presence of one or more DNA repair pathways or components thereof, the Cas-CLOVER method interrupts gene expression or modifies the genomic sequence by inserting, deleting, or substituting one or more base pairs. DSBs in cells can be repaired via non-homologous end joining (NHEJ, a mechanism that typically causes insertions or deletions (insertion/deletion) in DNA). Insertions/deletions typically induce frameshifts, resulting in loss-of-function alleles; for example, by inducing premature stop codons within the open reading frame (ORF) of a target gene. According to some implementations, the result is a loss-of-function mutation within a target immune checkpoint gene.

Alternatively, instead of NHEJ, DSB induced by the Cas-CLOVER system can be repaired via homology-directed repair (HDR). Although NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to produce specific nucleotide changes ranging from a single nucleotide change to a large insertion range. According to some implementations, HDR is used to gene edit immune checkpoint genes by delivering a DNA repair template containing the desired sequence to a TIL equipped with a Cas-CLOVER system. The repair template preferably contains the desired edit immediately upstream and downstream of the target gene, as well as other homologous sequences (often referred to as left and right homologous arms).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the Cas-CLOVER method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF1. 0A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, I L6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via the Cas-CLOVER method and which can be used according to embodiments of the invention are described in WO2019126578, US2017/0107541, US2017/0114149, US2018/0187185, and U.S. Patent No. 10,415,024, the contents of which are incorporated herein by reference in their entirety. Resources for performing the Cas-CLOVER method, such as CLOVER mRNA and Cas-CLOVER mRNA constructs, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system regulating the expression of at least one checkpoint protein in multiple cells of a second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culture for about 1 to 3 days to obtain the second TIL population, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system inhibiting the expression of at least one checkpoint protein in multiple cells of the second TIL population.

IV. Gene Expression Methods

In some embodiments, the method of genetically modifying TIL populations to regulate protein expression may optionally include a step of stabilizing and incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying TIL populations includes a viral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a gamma-retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adenovirus transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adeno-associated virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a herpes simplex virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a poxvirus transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step, including lentiviral transduction using human immunodeficiency virus (HIV) (including HIV-1). Lentiviral transduction systems and other suitable viral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat’l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71, and U.S. Patents 5,350,674, 5,585,362, and 6,627,442, the disclosures of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16; Hawley et al., Gene Ther. 1994, 1, 136-38; the contents of which are incorporated herein by reference.

1. piggyBac method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs by a piggyBac method (e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases). According to a particular embodiment, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population. Alternatively, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population and optionally induce the enhancement of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. In some embodiments, at least one immunomodulatory composition comprises an immunomodulator fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and CD40 agonists (e.g., CD40L or agonistic CD40 binding domains). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and CD40 agonists. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and CD40 agonists.

The piggyBac transposon is a mobile genetic element that efficiently translocates between the donor vector and the host chromosome. This system has virtually no cargo limitations and is completely reversible, leaving no footprint in the genome after excision. The piggyBac transposon/transposase system consists of transposases that recognize piggyBac-specific inverted terminal repeats (ITRs) located flanking the transposon cassette. The transposases excise the transposable element to integrate it into a TT/AA chromosomal locus that preferentially resides in euchromatin regions of the mammalian genome (Ding et al., 2005; and Bradley 2007; Wilson et al., 2007; Wang et al., 2008; Li et al., 2011).

Exemplary piggyBac systems include those described in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac system comprises a transposon/transposase system.

In the implementation scheme, the piggyBac method includes delivering to the TIL a (a) nucleic acid or amino acid sequence containing a sequence encoding a transposase and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon.

In one embodiment, the sequence encoding the transposase is an mRNA sequence. In another embodiment, the sequence encoding the transposase is a DNA sequence. In yet another embodiment, the DNA sequence is a cDNA sequence. In yet another embodiment, the sequence encoding the transposase is an amino acid sequence. The protein Superpiggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA.

Transposons/transposases

Exemplary transposon/transposase systems include, but are not limited to, piggyBac transposon and transposase, Sleeping Beauty transposon and transposase, Helraiser transposon and transposase, and Tol2 transposon and transposase.

The piggyBac transposon recognizes the transposon-specific inverted terminal repeat (ITR) at the end of the transposon and moves the contents between the ITRs to the TTAA chromosomal locus. The piggyBac transposon system has no effective load limitation on the gene of interest that can be included between the ITRs. In embodiments, the transposon is a piggyBac transposon or a piggyBac-like transposon.

Examples of piggyBac and piggyBac-like transposases and transposons include, for example, those disclosed in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is overactive. An overactive piggyBac or piggyBac-like transposase is a transposase that is more active than its naturally occurring variants. In embodiments, the overactive piggyBac or piggyBac-like transposase is isolated from or derived from the silkworm (Bombyxmori). A list of overactive amino acid substitutions is available in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is an integration-deficient type. In embodiments, an integration-deficient piggyBac or piggyBac-like transposase is a transposase capable of cleaving its corresponding transposon but integrating the cleaved transposon at a lower frequency than the corresponding wild-type transposase. A list of defective amino acid substitutions can be found in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

In the embodiments, the piggyBac or piggyBac-like transposon can be inserted into the 5'-TTAT-3 sequence within the target nucleic acid via a piggyBac or piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or SuperpiggyBac ^™ (SPB) transposase. In the embodiments, and particularly in embodiments where the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

The Sleeping Beauty (SB) transposon is translocated into the target genome via a Sleeping Beauty transposase that recognizes an ITR, and moves the contents between ITRs to the TA chromosomal locus. In an embodiment, the transposon is a Sleeping Beauty transposon. In an embodiment, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In an embodiment, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase.

The Helraiser transposon is translocated by the Helitron transposase. Unlike other transposases, the Helitron transposase does not contain an RNase-H-like catalytic domain, but instead contains a RepHel motif consisting of a replication initiation domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH nuclease superfamily. In embodiments, the transposon is the Helraiser transposon. In embodiments of the Helraiser transposon sequence, the transposase is flanked by left and right terminal sequences, referred to as the LTS and RTS. In embodiments, these sequences terminate with a conserved 5'-TC/CTAG-3' motif. In embodiments, a 19 bp palindromic sequence with the potential to form a hairpin termination structure is located 11 nucleotides upstream of the RTS and contains the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG. In embodiments, and particularly in embodiments where the transposon is the Helraiser transposon, the transposase is the Helitron transposase.

The Tol2 transposon can be isolated from or derived from the genome of the killifish and can be similar to transposons of the hAT family. An exemplary Tol2 transposon of this disclosure is encoded by a sequence comprising approximately 4.7 kilobases and contains a gene encoding a Tol2 transposase, said gene containing four exons. In embodiments, the transposon is a Tol2 transposon. In some embodiments of the methods of this disclosure, and particularly in those embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

In some embodiments, the vector comprises a recombinant and non-naturally occurring DNA sequence encoding a transposon. In some embodiments, the vector comprises DNA of any form, and wherein the vector comprises at least 100 nucleotides (nt), 500 nt, 1000 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6500 nt, 7000 nt, 7500 nt, 8000 nt, 8500 nt, 9000 nt, 9500 nt, 10,000 nt, or any number of nucleotides in between. In some embodiments, the vector comprises single-stranded or double-stranded DNA. In some embodiments, the vector comprises circular DNA. In some embodiments, the vector is a plasmid vector, a nanoplasmid vector, or a small loop. In some embodiments, the vector comprises linear or linearized DNA. In some embodiments, the vector is a double-stranded doggybone ^™ DNA sequence.

In the implementation scheme, the recombinant and non-naturally occurring DNA sequences encoding transposons also include sequences encoding one or more immune checkpoint genes.

In the implementation scheme, the nucleic acid sequence encoding the transposase is a DNA sequence, and the amount of the DNA sequence encoding the transposase and the DNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the DNA sequences encoding transposases and the DNA sequences encoding transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation scheme, the nucleic acid sequence encoding the transposase is an RNA sequence, and the amount of the RNA sequence encoding the transposase and the RNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the RNA sequences encoding transposases and transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation, the TIL is further modified by a second gene-editing tool, including but not limited to those described herein. In the implementation, the second gene-editing tool may include a piggyBac transposase that is only excised to re-excise the inserted sequence or any portion thereof. For example, a piggyBac transposase that is only excised can be used to "re-excise" a transposon.

According to some embodiments, the piggyBac system comprises a transposon/transposase system, wherein the transposase recognizes ITRs located flanking a transposon cassette containing a cargo encoding one or more immune checkpoint genes, and excises transposable elements to integrate it into a TT/AA chromosomal site, resulting in genomic insertion of the transposon cassette and expression of one or more immune checkpoint genes. According to some embodiments, the cargo encodes two or more immune checkpoint molecules.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the piggyBac method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD), and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using the piggyBac method and which can be used according to embodiments of the invention are described in WO2019/046815, WO2015006700, WO2010085699, WO2010099301, WO2010099296, WO2006122442, WO2001081565, and WO1998040510, the contents of which are incorporated herein by reference in their entirety.

Resources for executing the piggyBac method, such as plasmids for expressing transposons/transposases, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a piggyBac system, wherein the at least one gene editor system affects the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population and regulates the expression of at least one checkpoint protein in multiple cells of the second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) The second TIL population is obtained by stimulating it with OKT-3 and culturing it for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivery of at least one gene editor system comprising the piggyBac system, said gene editor system influencing the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population. In some embodiments, the at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

In some embodiments, methods for genetically modifying TIL populations include using non-viral techniques, such as the piggybac method (e.g., piggybac transposons and transposases or piggybac-like transposons and transposases). In some embodiments, the method includes delivering to the TIL: (a) a nucleic acid or amino acid sequence containing a sequence encoding a transposase; and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an mRNA sequence. The mRNA sequence encoding the transposase can be generated in vitro. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is a DNA sequence. The DNA sequence encoding the transposase can be generated in vitro. The DNA sequence may be a cDNA sequence. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an amino acid sequence. The amino acid sequence encoding the transposase can be generated in vitro. A protein super piggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA. In some embodiments, the transposon is a piggybac transposon or a piggybac-like transposon. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In some embodiments, and particularly those in which the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In some embodiments, the piggyBac transposase comprises the amino acid sequence containing SEQ ID NO:14487 of WO2019046815. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or Super piggyBac ^™ (SPB) transposase. In some embodiments, and particularly those in which the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence. In some embodiments of the methods disclosed herein, the transposase is a piggyBac ^™ (PB) transposase. piggyBac (PB) transposases may contain or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to the following sequences:

In some embodiments of the methods disclosed herein, the transposon is a Sleeping Beauty transposon. In some embodiments of the methods disclosed herein, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase. In some embodiments, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween the following sequences:

In some embodiments, including those in which the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof, identity with the following sequences:

V.Gen 2TIL Manufacturing Process

Figures 1 and 2 depict an exemplary TIL process family called Gen 2 (also known as process 2A) containing some of these features. An implementation of Gen 2 is shown in Figure 2.

As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs to enhance their metabolic activity and thus improve relative health status prior to transplantation into a patient, as well as methods for testing said metabolic health status. As generally outlined herein, TILs are typically obtained from patient samples and manipulated to amplify their number prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below.

In some implementations, TILs can be cryopreserved. After thawing, they can also be restimulated before being infused into the patient to increase their metabolism.

In some embodiments, the first amplification (including a process called pre-REP and the process shown as step B in Figures 1 and 36) is shortened to 3 to 14 days, and the second amplification (including a process called REP and the process shown as step D in Figure 1 or 36) is shortened to 7 to 14 days, as discussed in detail below, in the embodiments, and in the accompanying drawings. In some embodiments, the first amplification (e.g., the amplification described as step B in Figure 1 or 36) is shortened to 11 days, and the second amplification (e.g., the amplification described as step D in Figure 1 or 36) is shortened to 11 days. In some embodiments, the combination of the first and second amplifications (e.g., the amplification described as steps B and D in Figure 1 or 36) is shortened to 22 days, as discussed in detail below, in the embodiments, and in the accompanying drawings.

The "steps" denoted as A, B, C, etc. in the following text refer to Figure 1 or Figure 36 and certain embodiments described herein. The order of the steps in the following text and Figures 1 and 36 is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, repetitions of steps, and/or omissions of steps.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples and then expanded into larger populations for further manipulation as described herein, optionally cryopreservation, restimulation as outlined herein, and optionally evaluation of phenotypic and metabolic parameters as indicators of TIL health status.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multifocal sampling is used. In some embodiments, surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells include multifocal sampling (i.e., obtaining samples from one or more tumor sites and/or locations in a patient, as well as from one or more tumors at the same location or closely adjacent to each other). Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be lung tissue. In some embodiments, the applicable TIL is obtained from non-small cell lung cancer (NSCLC). Solid tumors can be skin tissue. In some embodiments, the applicable TIL is obtained from melanoma.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as HBSS.

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is a 10X working stock solution at a concentration of 100 mg/mL.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is a 10X working stock solution of 10,000 IU/mL.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is a 10X working stock solution at a concentration of 10 mg/mL.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatic tumor digests and tumor fragments obtained by digesting or fragmenting tumor samples obtained from patients.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as shown in Figure 1 or Figure 36), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor is 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor is 2mm × 2mm × 2mm. In some embodiments, the tumor is 3mm × 3mm × 3mm. In some embodiments, the tumor is 4mm × 4mm × 4mm.

In some embodiments, the tumor is removed to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of necrotic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of adipose tissue in each mass.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be used to remove these cells.

In some implementations, the cell suspension collected prior to the first amplification step is referred to as the “primary cell population” or “freshly collected” cell population.

In some embodiments, the cells may optionally be frozen after sample collection and cryopreserved before proceeding to the amplification described in step B (which is described in further detail below and in examples in Figures 1 and 36 and Figure 8).

1. Pleural effusion T cells and TIL

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may originate from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may originate from secondary metastatic cancer cells originating from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural transudate. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments where the disclosed methods utilize pleural fluid, ascites fluid or other cystic fluids containing TILs can be used to perform the same methods to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to further processing steps. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to further processing steps. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of viable TILs. If retained in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in further processing steps of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

B. Step B: First Amplification

In some embodiments, the method of the present invention achieves the acquisition of young TILs, which, compared to older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to the subject/patient), enable increased replication cycles at the time of administration to the subject/patient and thus provide additional therapeutic benefits. Characteristics of young TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32: 415-423; Robbins et al., J. Immunol. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, which are each incorporated herein by reference.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (connection region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 1 or Figure 36. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method referred to as Process 1C, as exemplified in Figures 5 and/or 6. In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased.

Following the dissection or digestion of the tumor fragment as described in step A of, for example, Figure 1 or Figure 36, the resulting cells are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 3 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 7 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for about 11 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells.

In some implementations, TIL amplification may be performed using the initial host TIL amplification steps described below and herein (e.g., the steps described in step B of Figure 1 or Figure 36, which may include a process called pre-REP), followed by a second amplification step described below and herein (step D, including a process called rapid amplification protocol (REP) step), followed by optional cryopreservation, and then a second step D described below and herein (including a process called restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic traits and metabolic parameters as described herein.

In TIL culture, the culture is initiated in a 24-well plate, such as using a Costar 24-well cell culture cluster, with a flat bottom (Corning Incorporated, Corning, NY). Each well is seeded with 2 mL of complete culture medium (CM) containing 1 × ^10⁶ tumor digested cells or one tumor fragment and IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digestion cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plate are incubated in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days.

After preparing the tumor fragment, the resulting cells (i.e., the fragment) are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum (or, in some cases, as outlined herein, in the presence of an APC cell population) and 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ primary TIL cells. In some embodiments, the growth medium contains IL-2 or a variant thereof during the initial expansion. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20-30 × ^10⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains IL-2 at concentrations between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL.

In some embodiments, the first amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the first amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the cell culture medium contains an anti-CD3 agonist antibody, such as OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, OKT-3 is present in the cell culture medium at the start of the first amplification (day 0). In some embodiments, OKT-3 is added to the cell culture medium at any time during the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 1 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 2 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 3 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 4 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 5 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 6 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 7 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 8 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 9 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 10 of the first amplification. In some implementations, OKT-3 is added to the cell culture medium on day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 1 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 3 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 6 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 7 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 8 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 9 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 10 and day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 2 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 3 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 4 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 5 of the first amplification.

In some implementations, OKT-3 is added once or more between day 5 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 6 of the first amplification.

In some implementations, OKT-3 is added once or more between day 6 and day 7 of the first amplification.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digest cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plates are cultured in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days. In some embodiments, CM is CM1 as described in the examples, see Example 1. In some embodiments, the first amplification is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 3-14 days, as discussed in the embodiments and figures. In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 7 to 14 days, as discussed in the embodiments and shown in FIG. 4 and FIG. 5, and includes amplification such as that described in step B of FIG. 1 or FIG. 36. In some embodiments, the first amplification in step B is shortened to 10-14 days. In some embodiments, the first amplification is shortened to 11 days, as discussed in the amplification described in step B of FIG. 1 or FIG. 36.

In some embodiments, the first TIL amplification can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the first TIL amplification can be performed for 1 to 14 days. In some embodiments, the first TIL amplification can be performed for 2 to 14 days. In some embodiments, the first TIL amplification can be performed for 3 to 14 days. In some embodiments, the first TIL amplification can be performed for 4 to 14 days. In some embodiments, the first TIL amplification can be performed for 5 to 14 days. In some embodiments, the first TIL amplification can be performed for 6 to 14 days. In some embodiments, the first TIL amplification can be performed for 7 to 14 days. In some embodiments, the first TIL amplification can be performed for 8 to 14 days. In some embodiments, the first TIL amplification can be performed for 9 to 14 days. In some embodiments, the first TIL amplification can be performed for 10 to 14 days. In some embodiments, the first TIL amplification can be performed for 11 to 14 days. In some embodiments, the first TIL amplification can proceed for 12 to 14 days. In some embodiments, the first TIL amplification can proceed for 13 to 14 days. In some embodiments, the first TIL amplification can proceed for 14 days. In some embodiments, the first TIL amplification can proceed for 1 to 11 days. In some embodiments, the first TIL amplification can proceed for 2 to 11 days. In some embodiments, the first TIL amplification can proceed for 3 to 11 days. In some embodiments, the first TIL amplification can proceed for 4 to 11 days. In some embodiments, the first TIL amplification can proceed for 5 to 11 days. In some embodiments, the first TIL amplification can proceed for 6 to 11 days. In some embodiments, the first TIL amplification can proceed for 7 to 11 days. In some embodiments, the first TIL amplification can proceed for 8 to 11 days. In some embodiments, the first TIL amplification can proceed for 9 to 11 days. In some embodiments, the first TIL amplification can proceed for 10 to 11 days. In some embodiments, the first TIL amplification can proceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the first amplification period. In some embodiments, the first amplification period, including, for example, during step B of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, as discussed in the examples and figures, the first amplification process (including a process called pre-REP; for example, step B according to Figure 1 or Figure 36) is shortened to 3 to 14 days. In some embodiments, the first amplification in step B is shortened to 7 to 14 days. In some embodiments, the first amplification in step B is shortened to 10 to 14 days. In some embodiments, the first amplification is shortened to 11 days.

In some embodiments, the first amplification is performed in a closed-system bioreactor, such as step B according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

1. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Transition from the first amplification to the second amplification

In some cases, the host TIL population obtained from the first amplification, including, for example, the TIL population obtained from step B as shown in Figure 1 or Figure 36, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first amplification (referred to as the second TIL population) can undergo a second amplification (which may include an amplification sometimes referred to as REP) and then be cryopreserved as discussed below. Similarly, where the genetically modified TILs are intended for therapeutic use, the first TIL population (sometimes referred to as the host TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be genetically modified before amplification or after the first amplification and before the second amplification for use in a suitable treatment.

In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is stored until a phenotypic analysis for selection is performed. In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is not stored and proceeds directly to the second amplification. In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 10 days after fragmentation. In some implementations, the transition from the first amplification to the second amplification occurs approximately 7 to 14 days after fragmentation. In other implementations, the transition from the first amplification to the second amplification occurs approximately 14 days after fragmentation.

In some embodiments, the transition from the first amplification to the second amplification occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 1 to 14 days after fragmentation. In some embodiments, the first TIL amplification may occur 2 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 5 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 7 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after fragmentation. In some embodiments, the transition occurs 9 to 14 days after fragmentation. In some embodiments, the transition occurs 10 to 14 days after fragmentation. In some embodiments, the transition occurs 11 to 14 days after fragmentation. In some embodiments, the transition occurs 12 to 14 days after fragmentation. In some embodiments, the transition occurs 13 to 14 days after fragmentation. In some embodiments, the transition occurs 14 days after fragmentation. In some embodiments, the transition occurs 1 to 11 days after fragmentation. In some embodiments, the transition occurs 2 to 11 days after fragmentation. In some embodiments, the transition occurs 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 11 days after fragmentation. In some embodiments, the transition occurs 5 to 11 days after fragmentation. In some embodiments, the transition occurs 6 to 11 days after fragmentation. In some embodiments, the transition occurs 7 to 11 days after fragmentation. In some embodiments, the transition occurs 8 to 11 days after fragmentation. In some embodiments, the transition occurs 9 to 11 days after fragmentation. In some embodiments, the transition occurs 10 to 11 days after fragmentation. In some embodiments, the transition occurs 11 days after fragmentation.

In some embodiments, TILs are not stored after the first amplification and before the rapid second amplification, and the TILs undergo the second amplification directly (e.g., in some embodiments, no storage occurs during the transition from steps B to D as shown in Figure 1 or Figure 36). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from the first amplification (the second TIL population) undergo the second amplification directly without a transition period.

In some embodiments, the transition from the first amplification to the second amplification (e.g., step C according to Figure 1 or Figure 36) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

According to this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from MiltenyiBiotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulation of TIGIT expression and downregulation of one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step also includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Second amplification

In some embodiments, the number of TIL cell populations is expanded after collection and initial host treatment, for example, after steps A and B and a transition referred to as step C (as indicated in Figure 1 or Figure 36). This further expansion is referred to herein as a second expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP); and the process indicated in step D of Figure 1 or Figure 36. The second expansion is typically performed in a breathable container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 1 or Figure 36) may be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL amplification may be performed for about 7 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 8 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 9 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 10 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 11 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 12 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 13 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 14 days.

In some embodiments, the second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the procedures indicated in step D of Figure 1 or Figure 36). For example, TILs can be rapidly amplified using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as about 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (available from BioLegend, San Diego, CA). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation to the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of: urinumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, the second amplification period, including, for example, during step D of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step D of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, during rapid expansion and/or secondary expansion, the ratio of TILs to PBMCs and/or antigen-presenting cells is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:100 and 1:200.

In some implementations, REP and/or secondary amplification are performed in culture flasks, where the host TIL is mixed with 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. The medium is replaced (typically 2/3 replaced with fresh medium via aspiration) until the cells are transferred to a replacement growth chamber. The replacement growth chamber comprises G-REX culture flasks and aeration containers, as discussed more fully below.

In some embodiments, the second amplification (which may include a process referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second amplification is shortened to 11 days.

In some embodiments, the REP and/or second amplification can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or aeration dishes (G-REX culture flasks). In some embodiments, the second amplification (including amplification referred to as rapid amplification) is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs suspended in 150 mL of medium can be added to each T-175 culture flask. The TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The T-175 culture flasks can be incubated at 37°C and 5% _CO₂ . Half of the medium can be replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some implementations, on day 7, cells from two T-175 culture flasks can be combined in a 3L bag, and 300 mL of AIM V with 5% human AB serum and 3000 IU/mL IL-2 can be added to 300 mL of TIL suspension. The cell count in each bag is counted daily or every two days, and fresh culture medium is added to maintain the cell count between 0.5 and 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (which may include an amplification referred to as REP, as well as the amplification mentioned in step D of Figure 1 or Figure 36) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing, New Brighton, MN, USA) with a 100 cm aerated silica bottom. 5 × ^10⁶ or 10 × ^10⁶ TILs can be cultured together with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). The G-REX-100 culture flask can be incubated at 37°C in 5% _CO₂ . On day 5, 250 mL of the supernatant can be removed and transferred to a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. TIL precipitate can be resuspended in 150 mL of fresh culture medium containing 5% human AB serum and 3000 IU/mL IL-2, and added back to the initial G-REX-100 culture flask. When TILs are serially amplified in G-REX-100 culture flasks, on day 7, TILs from each G-REX-100 flask can be resuspended in 300 mL of culture medium present in each flask, and the cell suspension can be aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 can be added to each culture flask. The G-REX-100 culture flasks can be incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 can be added to each G-REX-100 culture flask. Cells can be harvested on day 14 of culture. In some implementations, the process uses different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different number of repetitions.

In some embodiments, the second amplification (including an amplification termed REP) is performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some embodiments, two-thirds of the medium is replaced with fresh medium by aspiration. In some embodiments, the alternative growth chamber comprises a G-REX culture flask and a ventilated container, as discussed more fully below.

In some embodiments, a second amplification (including an amplification referred to as REP) is performed, and the process also includes a step of selecting the TIL for superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability assays may be performed after a second amplification (including amplification known as REP amplification) using standard assays known in the art. For example, a trypan blue exclusion assay may be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample may be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, the second amplification of TILs (including amplification referred to as REP) can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., 2008, Jimmunother., 31, 742-751, and Dudley et al., 2003, Jimmunother., 26, 332-342) or aeration G-REX culture flasks. In some embodiments, the second amplification is performed using culture flasks. In some embodiments, the second amplification is performed using aeration G-REX culture flasks. In some embodiments, the second amplification is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs are suspended in approximately 150 mL of culture medium and added to each T-175 culture flask. TILs were cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs used as "feeder" cells, and the cells were cultured in a 1:1 mixture (50/50 medium) of CM and AIM-V supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. T-175 culture flasks were incubated at 37°C and 5% _CO2 . In some embodiments, half of the medium was replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 culture flasks were pooled in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 was added to 300 mL of TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh culture medium can be added to maintain the cell count between approximately 0.5 and approximately 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (including an amplification referred to as REP) is performed in a 500 mL volumetric flask (G-REX-100, Wilson Wolf) with a 100 ^cm² permeable silica bottom. Approximately 5 × ^10⁶ or 10 × ^10⁶ TILs are incubated with irradiated allogeneic PBMCs at a 1:100 ratio in 400 mL of 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The G-REX-100 flask is incubated at 37°C and 5% _CO₂ . In some embodiments, on day 5, 250 mL of the supernatant is removed and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL precipitate is then resuspended in 150 mL of fresh 50/50 medium with 3000 IU/mL IL-2 and added back to the initial G-REX-100 flask. In the embodiment of continuous TIL amplification in G-REX-100 culture flasks, on day 7, the TIL from each G-REX-100 flask is resuspended in 300 mL of culture medium present in each flask, and the cell suspension is aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 is added to each culture flask. The G-REX-100 culture flasks are incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 is added to each G-REX-100 culture flask. Cells are harvested on day 14 of culture. In some embodiments, the process is performed using different centrifugation speeds (400 g, 300 g, 200 g, for 5 minutes) and different number of repetitions.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some implementations, the second amplification medium (e.g., sometimes referred to as CM2 or second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the second amplification is performed in a closed-system bioreactor, such as step D according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve vertical scaling up of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve lateral scaling out of the culture by: (a) performing rapid or second amplification by culturing TILs in a first small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the TILs transferred from the first small-scale culture is cultured in a second small-scale culture for approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 5 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid or second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid or second amplification is performed for a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid or second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid or second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid or secondary amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid or secondary amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains the same components as the cell culture medium used for rapid or secondary expansion after splitting. In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains different components than the cell culture medium used for rapid or secondary expansion after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid or secondary expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

1. Feeder cells and antigen-presenting cells

In some embodiments, the second amplification procedure described herein (e.g., including the amplification described in step D of Figure 1 or Figure 36, and an amplification referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells placed into the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some implementations, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 50 × ^10⁶ TILs. In still other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 25 × ^10⁶ TILs.

In some embodiments, the second amplification procedure described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs in the second expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796A1, the disclosure of which is incorporated herein by reference.

Step E: Collect TIL

Cells can be collected after the second amplification step. In some embodiments, TILs are collected after one, two, three, four, or more amplification steps, as shown in, for example, Figure 1 or Figure 36. In some embodiments, TILs are collected after two amplification steps, as shown in, for example, Figure 1 or Figure 36.

TILs can be collected by any suitable and aseptic method, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the methods of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, collection, such as step E according to Figure 1 or Figure 36, is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, step E according to Figure 1 or Figure 36 is performed according to the process described herein. In some embodiments, the closed system is introduced under aseptic conditions via a syringe to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is employed.

In some embodiments, TIL is collected according to the methods described in the embodiments. In some embodiments, TIL between day 1 and day 11 is collected using the methods described in the steps mentioned herein, such as the day 11 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 24 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 22 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments.

Step F: Final preparation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 1 or Figure 36 and outlined in detail above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, TILs amplified using the APCs of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using the PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VI. Gen 3TIL Manufacturing Process

Without being limited by any particular theory, it is believed that the initiation of a first expansion to initiate T cell activation, followed by a rapid second expansion to enhance T cell activation, as described in the method of the present invention, allows for the preparation of expanded T cells that retain a "younger" phenotype, and therefore it is anticipated that the expanded T cells of the present invention will exhibit higher cytotoxicity against cancer cells compared to T cells expanded by other methods. In particular, it is believed that initiating T cell activation by exposure to anti-CD3 antibodies (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), as taught in the method of the present invention, and then enhancing it by subsequent exposure to additional anti-CD3 antibodies (e.g., OKT-3), IL-2, and APCs, limits or avoids the maturation of T cells in the culture medium, thereby producing a population of T cells with a less mature phenotype, which are less exhausted by culture expansion and exhibit higher cytotoxicity against cancer cells. In some implementations, the rapid second expansion step is broken down into multiple steps to achieve vertical scaling up of culture scale by: (a) performing rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the T cells from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container and culturing the T cells from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the T cells transferred from the first small-scale culture is cultured in the second small-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs. In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some implementations, after rapid expansion, multiple subpopulations contain a therapeutically effective amount of TIL. In some implementations, after rapid expansion, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some implementations, after rapid expansion, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, rapid amplification is performed over a period of approximately 1 to 5 days before being split into multiple steps. In some implementations, the splitting of rapid amplification occurs approximately day 1, day 2, day 3, day 4, or day 5 after the start of rapid amplification.

In some implementations, the split of rapid amplification occurs approximately day 8, 9, 10, 11, 12, or 13 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of rapid amplification occurs approximately day 10 after the start of the first amplification. In another exemplary implementation, the split of rapid amplification occurs approximately day 11 after the start of the first amplification.

In some implementations, rapid amplification is performed for a period of approximately 4 to 11 days after the split. In some implementations, rapid amplification is performed for a period of approximately 3, 4, 5, 6, 7, 8, 9, 10, or 11 days after the split.

In some embodiments, the cell culture medium used for rapid expansion before splitting contains the same components as the cell culture medium used for rapid expansion after splitting. In some embodiments, the cell culture medium used for rapid expansion before splitting contains different components than the cell culture medium used for rapid expansion after splitting.

In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, the fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

In some implementations, rapid second expansion is performed after the T cell activation achieved by initiating the first expansion begins to decrease, slow down, decline, or subside.

In some cases, rapid second expansion occurs when T cell activation, achieved through initiating the first expansion, has decreased by just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, or 47%. The percentages are 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 100%.

In some implementations, the rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

In some implementations, rapid second expansion occurs when T cell activation achieved by initiating the first expansion has decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46%. The percentages are 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after that.

In some cases, rapid second expansion occurs when T cell activation achieved through initiating first expansion has decreased to at most just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%. %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, the reduction in T cell activation achieved by initiating the first expansion is determined by a decrease in the amount of interferon-γ released by T cells in response to antigen stimulation.

In some implementations, the initial expansion of T cells occurs within a period of at most exactly 7 days or about 8 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or approximately 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, the initial expansion of T cells is carried out over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, rapid secondary expansion of T cells takes place over a period of at most exactly or about 11 days.

In some implementations, rapid secondary expansion of T cells is carried out within a period of at most exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, rapid secondary expansion of T cells is carried out over time periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 11 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 8 days, and the rapid second expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 9 days.

In some implementations, the initial expansion of T cells occurs over an 8-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 9 days.

In some implementations, the initial expansion of T cells occurs over a 7-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, T cells are tumor-infiltrating lymphocytes (TILs).

In some implementations, the T cells are bone marrow infiltrating lymphocytes (MIL).

In some implementations, the T cells are peripheral blood lymphocytes (PBLs).

In some implementations, the T cells are obtained from a donor with cancer.

In some implementations, T cells are TILs obtained from tumors removed from patients with cancer.

In some implementations, the T cells are MILs obtained from the bone marrow of patients with hematological malignancies.

In some implementations, the T cells are peripheral blood mononuclear cells (PBMCs) derived from a donor. In some implementations, the donor has cancer. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the donor has a tumor. In some implementations, the tumor is a liquid tumor. In some implementations, the tumor is a solid tumor. In some implementations, the donor has a hematological malignancy.

In some aspects of this disclosure, immune effector cells (e.g., T cells) can be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL separation). In a preferred aspect, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected via apheresis may be washed to remove plasma fractions and optionally placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations. In one aspect, T cells are separated from peripheral blood lymphocytes by lysing erythrocytes and, for example, by depleting monocytes through PERCOLL gradient centrifugation or by countercurrent centrifugation.

In some embodiments, the T cells are PBLs isolated from whole blood or lymphocyte-rich apheresis products from a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematologic malignancy. In some embodiments, PBL is isolated from whole blood or lymphocyte-rich apheresis products using a positive or negative selection method, i.e., removing PBL using markers of the T-cell phenotype (e.g., CD3+CD45+), or removing non-T-cell phenotype cells while leaving PBL. In other embodiments, PBL is separated by gradient centrifugation. After PBL is isolated from donor tissue, the initial amplification of PBL can be initiated according to the initial amplification step of any method described herein by seeding a suitable number of isolated PBLs (approximately 1 × ^10⁷ PBLs in some embodiments) into an initial amplification culture.

An exemplary TIL process containing some of these features, referred to as process 3 (also referred to herein as Gen 3), is depicted in FIG8 (in particular, for example, FIG8B and/or FIG8C and/or FIG8D) and FIG36, and some advantages of this embodiment of the invention compared to Gen 2 are described in FIG1, FIG2, FIG8, FIG30 and FIG31 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). Implementations of Gen 3 are shown in Figures 1, 8, 30, and 36 (particularly, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). Process 2A or Gen 2 is also described in U.S. Patent Publication No. 2018/0280436, which is incorporated herein by reference in its entirety. The Gen 3 process is also described in International Patent Publication WO 2020/096988.

As discussed and generally summarized herein, TILs are derived from patient samples and manipulated using a TIL amplification process described herein and referred to as Gen 3 to amplify their number before transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below. In some embodiments, TILs may be cryopreserved before or after amplification. After thawing, they may also be restimulated to increase their metabolism before infusion into a patient.

In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the process referred to herein as the Rapid Amplification Protocol (REP) and the process shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 7 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (in particular, e.g., Figure 8B and/or Figure 8C)) takes 1 to 7 days, and the rapid second amplification (including the process referred herein as rapid amplification protocol (REP), and the process shown as step D in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) takes 1 to 10 days, as discussed in detail below and in the embodiments and figures. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 9 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some embodiments, the combination of initiating the first amplification and rapid second amplification (e.g., amplification described in Figure 8 (particularly, e.g., Figure 8B and/or Figure 8C as steps B and D) lasts 14-16 days, as discussed in detail below and in the examples and figures. In particular, it is believed that certain embodiments of the invention include an initiating first amplification step, wherein TILs are activated by exposure to an anti-CD3 antibody (e.g., OKT-3) in the presence of IL-2 or by exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody (e.g., OKT-3). In some embodiments, the TILs activated in the initiating first amplification step as described above are a first TIL population, i.e., a primary cell population.

The “step” designations A, B, C, etc. in the following text are non-limiting examples with reference to Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and certain non-limiting embodiments described herein. The order of steps in the following text and Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, step repetitions, and/or step omissions.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples (“primary TILs”) or from circulating lymphocytes (e.g., peripheral blood lymphocytes, including peripheral blood lymphocytes with TIL-like characteristics), and are then expanded into larger populations for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotype and metabolic parameters as indicators of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be any type of cancer, including (but not limited to) breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including (but not limited to) squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the applicable TIL is obtained from malignant melanoma tumors because reports indicate that these tumors have particularly high levels of TIL.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. TILs are cultured from these fragments using enzymatically digested tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociating enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a certain amount of sterile buffer (e.g., HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is 100 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally, cell suspensions obtained from tumors are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as provided in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), the tumor is physically fragmented. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor fragments are 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor fragments are 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2mm × 2mm × 2mm. In some embodiments, the tumor fragment is 3mm × 3mm × 3mm. In some embodiments, the tumor fragment is 4mm × 4mm × 4mm.

In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue in each fragment. In some embodiments, the tumor fragmentation step is an in vitro or ex vivo method.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, the cell suspension prior to initiating the first amplification step is referred to as the “primary cell population” or “freshly acquired” or “freshly isolated” cell population.

In some embodiments, the cells may optionally be frozen after sample separation (e.g., after obtaining a tumor sample and/or after obtaining a cell suspension from a tumor sample) and cryopreserved before proceeding to the amplification described in step B, which is further described in detail below and illustrated in Figure 8 (particularly, for example, Figure 8B).

1. TIL derived from hollow needles/small biopsies

In some implementations, TILs are initially obtained from patient tumor samples (“primary TILs”) obtained through core needle biopsy or similar procedures and then expanded into a larger population for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters.

In some embodiments, patient tumor samples may be obtained using methods known in the art, typically via mini-biopsy, core needle biopsy, needle biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples may be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples may also be liquid tumors, such as those obtained from hematologic malignancies. In some embodiments, the sample may be derived from multiple small tumor samples or biopsies. In some embodiments, the sample may comprise multiple tumor samples from a single tumor from the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three, or four tumors from the same patient. In some embodiments, the sample may comprise multiple tumor samples from multiple tumors from the same patient. The solid tumor may be lung and/or non-small cell lung cancer (NSCLC).

Generally, cell suspensions obtained from the tumor core or fragments are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

In some implementations, if the tumor is metastatic and the primary lesion has been effectively treated/removed in the past, it may be necessary to remove a metastatic lesion. In some implementations, when available, minimally invasive methods are used to remove skin lesions or lymph nodes in the neck or axillary region. In some implementations, skin lesions are removed or small biopsies are taken. In some implementations, lymph nodes are removed or small biopsies are taken. In some implementations, the tumor is melanoma. In some implementations, the small biopsy of melanoma includes a mole or a portion thereof.

In some embodiments, a small biopsy is a perforated biopsy. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin surrounding a suspected mole. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin and removing a circular piece of skin. In some embodiments, a small biopsy is a perforated biopsy and removes a circular portion of the tumor.

In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed along with a small border of normal-looking skin.

In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy and only the most irregular portion of the mole or growth is collected. In some implementations, the small biopsy is an incisional biopsy, and the incisional biopsy is used when other techniques cannot be performed, such as when the suspected mole is very large.

In some implementations, the small biopsy is a lung biopsy. In some implementations, the small biopsy is obtained via bronchoscopy. Typically, bronchoscopy involves inserting a small instrument through the nose or mouth, down the throat, and into the bronchial passage under the patient's anesthesia, where the instrument is used to remove tissue. In some implementations, a transthoracic biopsy may be used when the tumor or growth cannot be reached via bronchoscopy. Typically, for a transthoracic biopsy, the patient is also under anesthesia and a needle is inserted directly through the skin to the suspected site to remove a small tissue sample. In some implementations, a transthoracic biopsy may require interventional radiology (e.g., using X-ray or CT scan-guided needle). In some implementations, the small biopsy is obtained via puncture biopsy. In some implementations, the small biopsy is obtained via endoscopic ultrasound (e.g., an endoscope with a light and placed in the esophagus via the mouth). In some implementations, the small biopsy is obtained surgically.

In some implementations, the small biopsy is a head and neck biopsy. In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy in which a small piece of tissue is removed from the area of abnormal appearance. In some implementations, if the abnormal area is easily accessible, a sample can be collected without hospitalization. In some implementations, if the tumor is deep inside the mouth or throat, the biopsy may need to be performed under general anesthesia in an operating room. In some implementations, the small biopsy is an excisional biopsy. In some implementations, the small biopsy is an excisional biopsy in which the entire area is removed. In some implementations, the small biopsy is a fine-needle aspiration (FNA). In some implementations, the small biopsy is a fine-needle aspiration (FNA) in which cells are extracted (aspirated) from the tumor or mass using a very fine needle attached to a syringe. In some implementations, the small biopsy is a perforated biopsy. In some implementations, the small biopsy is a perforated biopsy in which a suspicious area is removed using perforated forceps.

In some implementations, the small biopsy is a cervical biopsy. In some implementations, the small biopsy is obtained via colposcopy. Typically, the colposcopy method uses a light-assisted magnifying instrument (coloscope) attached to a binocular magnifying lens, which is then used to examine a small portion of the cervix. In some implementations, the small biopsy is a cervical conization/cone biopsy. In some implementations, a small biopsy is a cervical conization/cone biopsy, in which an outpatient procedure may be required to remove a larger piece of tissue from the cervix. In some implementations, in addition to aiding in diagnosis, a cone biopsy can also be used as initial treatment.

The term "solid tumor" refers to an abnormal mass of tissue that typically does not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The histological structure of a solid tumor includes interdependent tissue compartments, comprising the parenchyma (cancer cells) and supporting stromal cells dispersed within it, providing a supportive microenvironment.

In some embodiments, samples from the tumor are obtained in the form of fine-needle aspiration (FNA), core needle biopsy, or mini-biopsy (including, for example, perforated biopsy). In some embodiments, the sample is first placed in a G-REX-10. In some embodiments, when one or two core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-10. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-100. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-500.

FNA can be obtained from skin tumors, including, for example, melanoma. In some implementations, the FNA is obtained from a skin tumor, such as one from a patient with metastatic melanoma. In some cases, the patient with melanoma has previously undergone surgical treatment.

FNA can be derived from lung tumors, including, for example, NSCLC. In some implementations, the FNA is derived from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the NSCLC patient has previously undergone surgical treatment.

The TILs described herein may be obtained from FNA samples. In some cases, FNA samples are obtained or separated from the patient using fine-gauge needles ranging from 18 to 25 gauge. Fine-gauge needles may be 18, 19, 20, 21, 22, 23, 24, or 25 gauge. In some embodiments, the FNA sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

In some cases, the TILs described herein are obtained from core needle biopsy samples. In some cases, the core needle biopsy sample is obtained or separated from the patient using a surgical or medical needle in the range of 11 to 16 gauge. The needle may be 11, 12, 13, 14, 15, or 16 gauge. In some embodiments, the core needle biopsy sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some implementations, the TIL is not derived from tumor digest. In some implementations, the solid tumor core is not fragmented.

In some embodiments, the tumor digest is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically dissociated again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically dissociated a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether it is incubated at 37°C in 5% _CO2 for another 30 minutes. In some implementations, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, obtaining the first TIL population involves a multifocal sampling method.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as Hank's balance salt solution (HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some implementations, the enzyme stock solution may be varied, thus requiring verification of the concentration of the lyophilized stock solution and corresponding modification of the final amount of enzyme added to the digestion mixture.

In some embodiments, the enzyme mixture comprises approximately 10.2 μl of neutral protease (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

2. Pleural effusion T cells and TILs

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may be derived from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells derived from another organ (e.g., breast, ovary, colon, or prostate). In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural effusion. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments of the pleural fluid in the examples of the present invention, ascites fluid or other cyst fluid containing TILs can be used for the same method to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to the contact step. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to the contact step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of live TILs. If retained in untreated pleural fluid, the number of live TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in the contact step is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in the contact step of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

3. Methods for expanding peripheral blood lymphocytes (PBL) from peripheral blood.

PBL Method 1. In some embodiments of the invention, PBL is amplification using the methods described herein. In some embodiments of the invention, the method includes obtaining a PBMC sample from whole blood. In some embodiments, the method includes enriching T cells by using negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC. In some embodiments, the method includes enriching T cells by using magnetic bead-based negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC.

In some embodiments of the present invention, PBL method 1 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. T cells are isolated using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

PBL Method 2. In some embodiments of the invention, PBL is amplified using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells from the PBMCs are enriched by incubating the PBMCs at 37°C for at least three hours and then separating non-adhesive cells.

In some embodiments of the present invention, PBL method 2 is performed as follows: On day 0, the frozen PMBC sample is thawed, and PBMC cells are seeded at 6 million cells per well in 6-well plates of CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, the non-adherent cells (which are PBL) are removed and their number is counted.

PBL Method 3. In some embodiments of the invention, PBL is amplified using PBL Method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection and T cells are selected using a non-CD19+ fraction of the PBMC sample using negative selection.

In some embodiments of the present invention, PBL method 3 is performed as follows: On day 0, frozen PBMCs derived from peripheral blood are thawed and their number is counted. CD19+ B cells are sorted using a CD19 multi-sorting human kit (Miltenyi Biotec). In the non-CD19+ cell fraction, T cells are purified using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

In some embodiments, PBMCs are isolated from whole blood samples. In some embodiments, PBMC samples are used as starting material for amplifying PBL. In some embodiments, the samples are cryopreserved prior to the amplification process. In other embodiments, fresh samples are used as starting material for amplifying PBL. In some embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human pan-T cell isolation kit and an LS column. In some embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art (e.g., CD19 negative selection).

In some embodiments of the invention, the PBMC sample is incubated for a period of time at a temperature required for effective identification of non-adhesive cells. In some embodiments of the invention, the incubation time is approximately 3 hours. In some embodiments of the invention, the temperature is approximately 37°C. The non-adhesive cells are then amplified using the process described above.

In some embodiments, PBMC samples are from subjects or patients who have optionally pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, tumor samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, PBMC samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or longer. In other embodiments, PBMCs are from patients currently receiving an ITK inhibitor regimen (e.g., ibrutinib).

In some implementations, PBMC samples are derived from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor and who are difficult to treat with a kinase inhibitor or an ITK inhibitor (e.g., ibrutinib).

In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment. In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment and have not been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or longer. In other embodiments, PBMCs are derived from patients who have previously been exposed to ITK inhibitors but have not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In some embodiments of the invention, on day 0, cells are selected for CD19+ and sorted accordingly. In some embodiments of the invention, antibody-bound beads are used for selection. In some embodiments of the invention, purified T cells are isolated from PBMCs on day 0.

In some embodiments of the present invention, for patients who have not been pretreated with ibrutinib or other ITK inhibitors, 10-15 mL of erythrocyte sedimentation rate (ESR) amber layer will produce approximately 5 × ^10⁹ PBMCs, which in turn will produce approximately 5.5 × ^10⁷ PBLs.

In some embodiments of the invention, for patients pretreated with ibrutinib or other ITK inhibitors, the amplification process will generate approximately 20 × ^10⁹ PBLs. In some embodiments of the invention, 40.3 × ^10⁶ PBMCs will generate approximately 4.7 × ^10⁵ PBLs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or obtained from any other method known in the art for obtaining PBMCs.

In some embodiments, the PBL is prepared using the method described in U.S. Patent Application Publication No. US 2020/0347350A1, the disclosure of which is incorporated herein by reference.

4. Methods for expanding bone marrow infiltrating lymphocytes (MILs) derived from bone marrow-derived PBMCs

MIL Method 3. In some embodiments of the invention, the method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+/CD33+/CD20+/CD14+ cells, and non-CD3+/CD33+/CD20+/CD14+ cell fractions are sonicated and a portion of the sonicated cell fractions are added back to the selected cell fractions.

In some embodiments of the present invention, MIL method 3 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. Cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using an S3e cell sorter (Bio-Rad). Cells are sorted into two fractions: immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and AML embryonic cell fraction (non-CD3+CD33+CD20+CD14+).

In some embodiments of the invention, PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow via apheresis, aspiration, biopsy, or other similar methods known in the art. In some embodiments, PBMCs are fresh. In other embodiments, PBMCs are cryopreserved.

In some embodiments of the invention, the MIL is expanded from 10-50 mL of bone marrow aspirate. In some embodiments of the invention, 10 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 20 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 30 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 40 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 50 mL of bone marrow aspirate is obtained from the patient.

In some embodiments of the invention, the number of PBMCs produced from about 10-50 mL of bone marrow aspirate is about 5 × ^10⁷ to about 10 × ^10⁷ PBMCs. In other embodiments, the number of PBMCs produced is about 7 × ^10⁷ PBMCs.

In some embodiments of the invention, approximately 5 × ^10⁷ to approximately 10 × ^10⁷ PBMCs produce approximately 0.5 × ^10⁶ to approximately 1.5 × ^10⁶ MILs. In some embodiments of the invention, approximately 1 × ^10⁶ MILs are produced.

In some embodiments of the invention, 12 × ^10⁶ PBMCs derived from bone marrow aspirate yield approximately 1.4 × ^10⁵ MILs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, bone marrow, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or from any other method known in the art for obtaining PBMCs.

In some embodiments, the MIL is prepared using the method described in U.S. Patent Application Publication No. US2020/0347350A1, the disclosure of which is incorporated herein by reference.

Step B: Initiating the first amplification

In some embodiments, the method of the present invention provides younger TILs that may offer additional therapeutic benefits compared to older TILs (i.e., TILs that have undergone further replication in multiple rounds prior to administration to the subject/patient). Characteristics of younger TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32, 415-423; Robbins et al., J. Immunother. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, each of which is incorporated herein by reference.

Following the segmentation or digestion of tumor fragments and/or tumor fragments as described in step A of, for example, Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) under conditions favorable to TIL growth but unfavorable to tumor and other cell growth. In some embodiments, IL-2, OKT-3, and feeder cells are added together with the tumor digest and/or tumor fragments at the start of culture (e.g., on day 0). In some embodiments, the tumor digest and/or tumor fragments are incubated in containers at a concentration of up to 60 fragments per container and with 6000 IU/mL IL-2. In some embodiments, culturing this primary cell population for several days, typically 1 to 8 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, culturing this primary cell population for several days, typically 1 to 7 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells.

In some implementations, TIL amplification may be performed using the steps described in step B of Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), which may include a process called pre-REP or initiating REP and which begins on day 0 and/or from the start of culture containing feeder cells), followed by a rapid second amplification (step D, including a process called the Rapid Amplification Protocol (REP) step) as described in step D below and here, followed by optional cryopreservation, and then a second step D as described below and here (including a process called the restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some implementations, the tumor fragments are between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.

In some embodiments, there are fewer or equal to 240 tumor fragments. In some embodiments, fewer or equal to 240 tumor fragments are placed in fewer or equal to 4 containers. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, fewer or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each container contains fewer or equal to 500 mL of culture medium. In some embodiments, the culture medium contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the culture medium contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains OKT-3. In some embodiments, the culture medium contains 30 ng/mL OKT-3 per container. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells ^per container.

After preparing the tumor fragment, the resulting cells (i.e., fragments of the primary cell population) are cultured in a medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions favorable to TIL growth but unfavorable to tumor and other cell growth, allowing TIL initiation and accelerated growth from day 0. In some embodiments, the tumor digest and/or tumor fragment are incubated with 6000 IU/mL IL-2, antigen-presenting feeder cells, and OKT-3. This primary cell population is cultured for several days, typically 1 to 8 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initial expansion period contains IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for several days, typically 1 to 7 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initiation of the first expansion comprises IL-2 or a variant thereof, along with antigen-presenting feeder cells and OKT-3. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20-30 ^{× 10⁶} IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × 10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the initial amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some embodiments, the initial expansion cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or about 8000 IU/mL IL-2.

In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial expansion medium contains approximately 200 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium also contains IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 2 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the initial cell culture medium for expansion contains OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, the initial amplification cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 6000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the initiation first expansion medium is referred to as "CM" (an abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In some embodiments, CM is CM1 as described in the examples. In some embodiments, the initiation of the first expansion is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initiation first expansion medium or initial cell culture medium or first cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (also referred to herein as feeder cells).

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific). In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some implementations, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisherScientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the specific culture medium described by reference to International PCT Publication WO/1998/030679, which is incorporated herein by reference, can be used in this invention. In that disclosure, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4+, Br, T, ^{Mn2 +} , P, Si4+, V5 ⁺ , Mo6 ⁺ , ^{Ni2 +} , Rb+, Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

Table 4: Concentration of Non-Trace Element Components

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) can be used in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 4 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 5 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG1 or FIG36 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 7 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 4 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8B and/or FIG8C), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 5 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 7 days, as discussed in the embodiments and figures.

In some embodiments, the initiation of first TIL amplification may be performed 1 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 1 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 7 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some implementations, the initiation of the first TIL amplification can be performed 7 days after fragmentation occurs and/or after the start of the first initiation amplification step.

In some implementations, the initial amplification of the TIL can take place for 1, 2, 3, 4, 5, 6, 7, or 8 days. In some implementations, the first TIL amplification can take place for 1 to 8 days. In some implementations, the first TIL amplification can take place for 1 to 7 days. In some implementations, the first TIL amplification can take place for 2 to 8 days. In some implementations, the first TIL amplification can take place for 2 to 7 days. In some implementations, the first TIL amplification can take place for 3 to 8 days. In some implementations, the first TIL amplification can take place for 3 to 7 days. In some implementations, the first TIL amplification can take place for 4 to 8 days. In some implementations, the first TIL amplification can take place for 4 to 7 days. In some implementations, the first TIL amplification can take place for 5 to 8 days. In some implementations, the first TIL amplification can take place for 5 to 7 days. In some implementations, the first TIL amplification can take place for 6 to 8 days. In some implementations, the first TIL amplification can take place for 6 to 7 days. In some implementations, the first TIL amplification can take place for 7 to 8 days. In some implementations, the first TIL amplification can take 8 days. In some implementations, the first TIL amplification can take 7 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, during the initiation of the first amplification, IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included, for example, during step B of the process described herein, according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step B.

In some embodiments, step B of initiating the first amplification (e.g., according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

1. Feeder cells and antigen-presenting cells

In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added during the initiation of the first amplification. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5-7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 6–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 6-7 of the initiation first amplification period. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 or 8 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 8 of the initiation of the first amplification.

In some embodiments, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8B) and those amplifications referred to as pre-REP or initiation REP) requires feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification and during the initiation of the first amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, 2.5 × ^10⁸ feeder cells are used during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per container during the initiation of the first amplification. In some embodiments, 2.5 × ^{10⁸ feeder cells are used per GREX-10} during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per GREX-100 during the initiation of the first amplification.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 has not increased compared to the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells. In other embodiments, the number of feeder cells required to initiate the first expansion is one-quarter, one-third, five-twelfths, or one-half of the number of feeder cells used for rapid second expansion.

In some embodiments, the culture medium for initiating the first expansion contains IL-2. In some embodiments, the culture medium for initiating the first expansion contains 6000 IU/mL IL-2. In some embodiments, the culture medium for initiating the first expansion contains antigen-presenting feeder cells. In some embodiments, the culture medium for initiating the first expansion contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium for initiating the first expansion contains OKT-3. In some embodiments, the culture medium contains 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells. In some embodiments, the culture medium comprises 500 mL of culture medium and ¹⁵ μg OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium comprises 500 mL of culture medium, 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the initiation of the first expansion procedure described herein requires an excess of feeder cells beyond the TIL during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs to initiate the first expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is also possible to initiate the first expansion of TILs using cytokines in combination with two or more of IL-2, IL-15, and IL-21 as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly beneficial for the generation of lymphocytes, and especially T cells as described therein. See, for example, Table 2.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives may be used in the culture medium during step B, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Initiating the transition from the first amplification to the rapid second amplification

In some cases, a population of primary TILs obtained from an initial first amplification (which may include amplification sometimes referred to as pre-REP), including, for example, a population of TILs obtained from step B as indicated in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), may undergo a rapid second amplification (which may include amplification sometimes referred to as a rapid amplification protocol (REP)) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs are intended for therapeutic use, an amplified TIL population from an initial first amplification or an amplified TIL population from a rapid second amplification may be genetically modified for appropriate treatment before the amplification step or after the initial first amplification and before the rapid second amplification.

In some implementations, the TIL obtained from step B as indicated in the initiation of the first amplification (e.g., Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is stored until the phenotype is determined for selection. In some embodiments, the TIL obtained from the initiation of the first amplification (e.g., step B as indicated in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is not stored and is directly subjected to rapid second amplification. In some embodiments, the TIL obtained from the initiation of the first amplification is not cryopreserved after the initiation of the first amplification and before rapid second amplification. In some embodiments, the transition from the initiation of the first amplification to the second amplification occurs approximately 2, 3, 4, 5, 6, 7, or 8 days after tumor fragmentation occurs and/or after the initiation of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 8 days after fragmentation and/or after the start of the first initiation amplification step.

In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1, 2, 3, 4, 5, 6, 7, or 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 6 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 6 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 8 days after fragmentation and/or the start of the first initiation amplification step.

In some embodiments, TILs are not stored after the initial first amplification and before the rapid second amplification, and the TILs undergo rapid second amplification directly (e.g., in some embodiments, they are not stored during the transition from step B to step D as shown in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from which the first amplification was initiated (the second TIL population) undergo rapid second amplification directly without a transition period.

In some embodiments, the transition from initiating the first amplification to the rapid second amplification (e.g., step C according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a GREX-10 or GREX-100. In some embodiments, the closed-system bioreactor is a single bioreactor. In some embodiments, the transition from initiating the first amplification to the rapid second amplification involves a longitudinal scaling up of the container size. In some embodiments, the initiation of the first amplification is carried out in a smaller container compared to the rapid second amplification. In some implementations, the initial amplification is performed in a GREX-100 and the rapid second amplification is performed in a GREX-500.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

In view of this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from Miltenyi Biotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or Figures 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing process also includes a quiescent step. According to some implementations, the quiescent step involves incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Rapid Second Amplification

In some implementations, the TIL cell population is further expanded in number after the collection and initiation of the first expansion (steps A and B) and the transition referred to as step C, as indicated in Figures 8 (in particular, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). This further amplification, referred to herein as rapid second amplification or rapid amplification, may include an amplification process commonly referred to in the art as a rapid amplification procedure (rapid amplification protocol or REP); and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Rapid second amplification is typically performed in a ventilated container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies. In some embodiments, the TIL is transferred to a larger volume container 1, 2, 3, or 4 days after the initiation of rapid second amplification (i.e., on days 8, 9, 10, or 11 of the overall Gen 3 process).

In some embodiments, the rapid second amplification of TIL (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 9 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 1 day after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 2 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 3 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 4 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 5 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 6 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 8 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 9 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 10 days after the initiation of rapid second amplification.

In some embodiments, the rapid second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the process indicated in step D of FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells, wherein feeder cells are added to a final concentration that is two, 2.4, 2.5, 3, 3.5, or 4 times the feeder cell concentration present in the initiating first amplification. For example, TILs can be rapidly expanded by stimulation with nonspecific T cell receptors in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as approximately 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Laritan, NJ or Miltenyi Biotech, Auburn, California), or UHCT-1 (available from BioLegend, San Diego, California). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation with the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL to 60 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3. In some embodiments, the cell culture medium comprises about 60 ng/mL of OKT-3. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium in the rapid second amplification contains 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 500 mL of culture medium and 6000 IU/mL IL-2, 30 μg of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium contains 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells. In some implementations, the culture medium in the rapid second amplification comprises 500 mL of culture medium per container and 6000 IU/mL IL-2, 30 μg OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, during the second amplification period, including, for example, during step D of the process described herein according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step D.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen-presenting cells in rapid expansion and/or secondary expansion is approximately 1:10, approximately 1:15, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:75, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs in rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some implementations, the ratio of TIL to PBMC in rapid amplification and/or secondary amplification is between 1:100 and 1:200.

In some implementations, the REP and/or rapid second amplification are performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 ng/mL OKT3 anti-CD3 antibody, and 6000 IU/mL IL-2 in 150 mL of culture medium, wherein the feeder cell concentration is at least 1.1-fold (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.8X, 2X, 2.1X, 2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, or 4.0X. The culture medium is replaced (usually by removing 2/3 of the used medium and replacing it with an equal volume of fresh medium) until the cells are transferred to a replacement growth chamber. The replacement growth chamber consists of G-REX culture flasks and aeration containers, as discussed in more detail below.

In some embodiments, the rapid second amplification (which may include a process referred to as the REP process) takes 7 to 9 days, as discussed in the examples and figures. In some embodiments, the second amplification takes 7 days. In some embodiments, the second amplification takes 8 days. In some embodiments, the second amplification takes 9 days.

In some embodiments, the second amplification (which may include an amplification referred to as REP, and the amplification mentioned in step D of Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA) with a 100 cm aerated silica bottom, 5 × ^10⁶ or 10 × ^10⁶ TILs can be taken together with PBMCs in 400 mL of supplemented 5% human AB serum at 3000 IU/mL Cells were cultured in a 50/50 medium containing IL-2 and 30 ng/mL anti-CD3 (OKT3). G-REX-100 culture flasks were incubated at 37°C in 5% _CO2 . On day 5, 250 mL of the supernatant was removed and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL precipitate was resuspended in 150 mL of fresh medium containing 5% human AB serum and 6000 IU/mL IL-2 and added back to the original GREX-100 culture flask. As TILs were continuously amplified in GREX-100 culture flasks, they could be transferred to larger culture flasks, such as GREX-500, on day 10 or 11. Cells could be collected on day 14. Cells could be collected on day 15. Cells could be collected on day 16. In some embodiments, the medium was replaced until the cells were transferred to a replacement growth chamber. In some embodiments, two-thirds of the culture medium is replaced by aspirating the used medium and replacing it with an equal volume of fresh medium. In some embodiments, alternative growth chambers include GREX culture flasks and aerated containers, as discussed more fully below. In some embodiments, the process employs different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different numbers of replicates.

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some implementations, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

In some embodiments, the specific culture media described in International Patent Application Publication No. WO 1998/030679 and U.S. Patent Application Publication No. US2002/0076747A1 (which are incorporated herein by reference) are applicable to the present invention. In the disclosures, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , ^{Cr3 +} , Ge4 ⁺ , Se4+, Br, T, ^Mn2+ , P, ^Si4+ , V5 ⁺ , Mo6 ⁺ , ^{Ni2 +, Rb+} , Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) is suitable for use in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, a rapid second amplification (including an amplification termed REP) is performed, and it further includes a step of selecting TILs with superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability can be determined using standard assays known in the art after a rapid second amplification (including amplification known as REP amplification). For example, a trypan blue exclusion assay can be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample can be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve vertical scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a first mini-culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the first mini-culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the TILs transferred from the first mini-culture is cultured in the second mini-culture for a period of approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 5 days; and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid second amplification is performed over a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid second amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid second amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid second expansion before splitting contains the same components as the cell culture medium used for rapid second expansion after splitting. In some embodiments, the cell culture medium used for rapid second expansion before splitting contains different components than the cell culture medium used for rapid second expansion after splitting.

In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

1. Feeder cells and antigen-presenting cells

In some embodiments, the rapid second amplification procedure described herein (e.g., including amplifications such as those described in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In other embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:10, approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 7.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, rapid second expansion requires twice the number of feeder cells of rapid second expansion. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 5 × ^10⁸ feeder cells. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 7.5 × ^10⁸ feeder cells. In other embodiments, the rapid second expansion requires twice (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X the number of feeder cells required to initiate the first expansion.

In some embodiments, the rapid second amplification procedure described herein requires an excess of feeder cells during the rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs. In some embodiments, PBMCs are added to the rapid second amplification at twice the concentration of PBMCs added to initiate the first amplification.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs during rapid second expansion.

2. Cytokines and other additives

The rapid second amplification method described herein typically uses a culture medium containing high doses of cytokines (particularly IL-2), as is known in the art.

Alternatively, it is possible to rapidly expand TILs using combinations of cytokines, as described in U.S. Patent Application Publication No. US2017/0107490 A1, which discloses the use of combinations of two or more IL-2, IL-15, and IL-21, the contents of which are incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having specific uses in many embodiments. The use of combinations of cytokines is particularly beneficial for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Furthermore, additives, such as peroxisome proliferator-activated receptor γ coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, may be used in the culture medium during step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

Step E: Collect TIL

Cells can be collected after the rapid second amplification step. In some embodiments, TILs are collected after one, two, three, four or more amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps (one initiating a first amplification and one rapid second amplification) provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

TILs can be collected in any suitable and aseptic manner, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the process of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some embodiments, step E according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) is performed according to the process described herein. In some embodiments, the closed system is introduced via a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described herein is employed.

In some embodiments, TILs are collected according to the methods described herein. In some embodiments, TILs between days 14 and 16 are collected using the methods described herein. In some embodiments, TILs are collected on day 14 using the methods described herein. In some embodiments, TILs are collected on day 15 using the methods described herein. In some embodiments, TILs are collected on day 16 using the methods described herein.

Step F: Final formulation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and as outlined above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, the TIL amplified using the methods of this disclosure is administered to the patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. The TIL amplified as disclosed herein can be administered via any suitable route known in the art. In some embodiments, the TIL is administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VII. Other Gen 2, Gen 3 and other TIL manufacturing process implementation plans

A. PBMC feeder cell ratio

In some embodiments, the culture medium used for the amplification methods described herein (see, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) includes an anti-CD3 antibody, such as OKT-3. The combination of anti-CD3 antibody and IL-2 induces T cell activation and cell division in TIL populations. This effect is seen in full-length antibodies as well as Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

In some implementations, the number of PBMC feeder cell layers is calculated as follows:

AT cell volume (diameter 10 μm): V = (4/3) ^πr³ = 523.6 ^μm³

B. The volume of G-REX-100(M) with a height of 40 μm (4 cells): V = (4/3) ^πr³ = 4 × ^{10¹² μm³}

C. Number of cells required to fill column B: 4 × ^10¹² μm³ ^{/ 523.6 μm³ =} 7.6 × ^{10⁸ μm³} × 0.64 = 4.86 × ^10⁸

D. The optimal number of cells to activate in 4D space: 4.86 × 10⁸ ^{/ 24} = 20.25 ^{× 10⁶}

E. Extrapolation to feeder cell and TIL numbers for G-REX-500: TIL: 100 × ^10⁶ and feeder cells: 2.5 × ^10⁹

In this calculation, the approximate number of mononuclear cells required to provide TIL activation using icosahedral geometry in a cylinder with a base of 100 ^cm² is used. The experimental result for T cell threshold activation derived from the calculation is approximately 5 × ^10⁸ , which is closely correlated with NCI experimental data, as described in Jin et al., J. Immunother. 2012, 35, 283-292. In (C), the multiplier (0.64) is the random filling density of the equivalent spheres, calculated by Jaeger and Nagel, Science, 1992, 255, 1523-3. In (D), the divisor 24 is the number of equivalent spheres or “Newton number” in 4-dimensional space that can contact similar objects, as described in Musin, Russ. Math. Surv., 2003, 58, 794-795.

In some embodiments, the number of exogenously supplied antigen-presenting feeder cells during the initiation of the first expansion is approximately half the number of exogenously supplied antigen-presenting feeder cells during the rapid second expansion. In some embodiments, the method includes initiating the first expansion in a cell culture medium containing approximately 50% fewer antigen-presenting cells compared to the cell culture medium used in the rapid second expansion.

In other embodiments, the number of exogenously supplied antigen-presenting feeder cells (APCs) during the rapid second amplification is greater than the number of exogenously supplied APCs during the initiation of the first amplification.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs during the rapid second amplification period to the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, or 2.5:1. 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately ¹ ×10⁸, 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, ^1.5 × ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8×10⁸, 1.9× ^10⁸ , 2 ^{×10⁸ ,} 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3×10⁸, 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^{10⁸ ,} 3.1× ^10⁸ , or 3.2×10⁸ ^. The number of exogenously supplied APCs during the rapid second amplification period is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × 10⁸, 3.8 × ^10⁸ , 3.9 × ^10⁸ , ^{4 × 10⁸ ,} 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^10⁸ , 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , ^4.9 × ^10⁸ , 5 × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^10⁸ , 5.3 × ^10⁸ , 5.4 ^{× 10⁸ .} 5.5× ^10⁸ , 5.6× ^10⁸ , 5.7×10⁸, 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3×10⁸, 6.4× ^10⁸ , ^6.5 × ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , ^7.2 × ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , 7.8× ^10⁸ , 7.9× ^10⁸ 8× ^10⁸ , 8.1× ^10⁸ , 8.2×10⁸, 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8×10⁸, 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , ^9.7 × ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or 1×10⁹ APCs .}

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ^1.5 × 10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ² × 10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is exactly or approximately 5 × ^10⁸ APCs.

In some embodiments, the number of APCs (including, for example, PBMCs) added on day 0 of the first expansion is approximately half the number of PBMCs added on day 7 of the first expansion (e.g., day 7 of the method). In some embodiments, the method includes adding antigen-presenting cells to a first TIL population on day 0 of the first expansion and adding antigen-presenting cells to a second TIL population on day 7, wherein the number of antigen-presenting cells added on day 0 is approximately 50% of the number of antigen-presenting cells added on day 7 of the first expansion (e.g., day 7 of the method).

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is greater than the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification.

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 2 × ^10⁶ APCs/ ^cm² to just or about 3 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density of just or about 2 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} Inoculate culture flasks with APCs/cm² at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4×10⁶ ^, or 4.5× ^{10⁶ .}

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 4.0 × ^10⁶ APCs/ ^cm² to just or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from the range of exactly or approximately 4.0 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenously supplied APCs in the rapid second amplification are at exactly or approximately 2.5 × ^10⁶ APCs/ ^cm² , 2.6 × 10⁶ APCs/ ^cm² , 2.7 × ^10⁶ APCs/ ^cm² , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , 3.2 × ^10⁶ , 3.3 × ^10⁶ , 3.4 × ^10⁶ , 3.5 × ^10⁶ , 3.6 × ^10⁶ , 3.7 × ^10⁶ , 3.8 × ^10⁶ , 3.9 × ^10⁶ , 4 × ^10⁶ , 4.1 × ^10⁶ , 4.2 × ^10⁶ , 4.3 × ^10⁶ , 4.4 × ^10⁶ , or 4.5 × ^{10⁶ .} 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8×10⁶, 4.9× ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4×10⁶, 5.5× ^10⁶ , ^{5.6×10⁶ ,} 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , 6× ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , ^6.3 × ^10⁶ , 6.4×10⁶, 6.5× ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , ^6.8 × ^10⁶ , 6.9× ^10⁶ , 7× ^10⁶ Inoculate the culture flasks at densities of 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4× ^10⁶ , or 7.5× ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} The APCs were seeded in culture flasks at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , or 4.5× ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for rapid second amplification were at exactly or approximately 2.5× ^10⁶ APCs/ ^cm² , 2.6× ^10⁶ APCs/ ^cm² , 2.7× ^10⁶ APCs/ ^cm² , 2.8× ^10⁶ , 2.9× ^10⁶ , 3× ^10⁶ , and 3.1×10⁶ ^APCs/cm². 3.2× ^10⁶ , 3.3× ^10⁶ , 3.4×10⁶, 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4×10⁶, 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , 4.5× ^10⁶ , 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8× ^10⁶ , ^4.9 × ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4× ^10⁶ , 5.5× ^10⁶ , ^5.6 × ^10⁶ APCs/cm² were inoculated into culture flasks at densities of 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , ⁶ × ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , 6.3×10⁶, 6.4× ^10⁶ , ^6.5 × ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , 6.8× ^10⁶ , 6.9×10⁶, ⁷ × ^10⁶ , 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4×10⁶, or 7.5× ^{10⁶ .}

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density selected ^from exactly or about 2.5 × ^10⁶ APCs/cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in culture flasks at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in culture flasks at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 2 × ^10⁶ APCs/ ^cm² to exactly or about 3 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied at the rapid second amplification are seeded in the culture flask at a density selected from exactly or about 4 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in the culture flask at a density of exactly or approximately 2 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density of exactly or approximately 4 × ^10⁶ APCs/ ^cm² .

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other implementations, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, etc. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1× ^10⁸ , 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4× ^10⁸ , 1.5× ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9× ^10⁸ , 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , or 3.1×10⁸ ^. The exogenous supply of APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × ^10⁸ , 3.8 × ^10⁸ , 3.9 × ^10⁸ , 4 × ^10⁸ , 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^{10⁸ ,} 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , 4.9 × ^10⁸ , ⁵ × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^{10⁸ . 8} , 5.3× ^10⁸ , 5.4× ^10⁸ , 5.5×10⁸, 5.6× ^10⁸ , 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , 7.2× ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^{10⁸ 8} , 7.8× ^10⁸ , 7.9×10⁸, ⁸ ×10⁸, 8.1× ^10⁸ , 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , 9.7× ^10⁸ , ^9.8 × ^10⁸ , 9.9× ^10⁸ , or 1× ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 1 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 1 × ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 7.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 2 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 4.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 5.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or about 5 × ^10⁸ APCs (including, for example, PBMCs).

In some embodiments, the number of APC (including, for example, PBMC) layers added on day 0 of the first amplification is approximately half the number of APC (including, for example, PBMC) layers added on day 7 of the rapid second amplification. In some embodiments, the method includes adding antigen-presenting cell layers to a first TIL population on day 0 of the first amplification and adding antigen-presenting cell layers to a second TIL population on day 7, wherein the number of antigen-presenting cell layers added on day 0 is approximately 50% of the number of antigen-presenting cell layers added on day 7.

In other implementations, the number of exogenously supplied APC (including, for example, PBMC) layers on day 7 of the rapid second amplification is greater than the number of exogenously supplied APC (including, for example, PBMC) layers on day 0 of the initiation of the first amplification.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about three cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1.5 to just or about 2.5 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about two cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers, and day 7 of the rapid second amplification occurs with an average thickness of just or approximately 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3. It occurs in the presence of layered APCs (including, for example, PBMCs) of 8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1 cell layer to just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 to just or about 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1, 2, or 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9, or 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is exactly or approximately 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from... Exactly or approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1 :4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.0 × ^10⁶ APCs/ ^cm² to about 4.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 2.5 × ^10⁶ APCs/ ^cm² to about 7.5 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.5 × ^10⁶ APCs/ ^cm² to about 3.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 3.5 × ^10⁶ APCs/ ^cm² to about 6.0 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 2.0 × ^10⁶ APCs/ ^cm² to about 3.0 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 4.0 × ^10⁶ APCs/ ^cm² to about 5.5 × ^10⁶ APCs/ ^cm² .

A. Optional cell culture medium components

1. Anti-CD3 antibody

In some embodiments, the culture medium used for the amplification methods described herein (including an amplification method known as REP, see, for example, Figures 1 and 8 (especially, for example, Figure 8B)) includes an anti-CD3 antibody. The combination of the anti-CD3 antibody and IL-2 induces T cell activation and cell division in a TIL population. This effect can be seen with both full-length antibodies and Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California). See Table 1.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California).

2,4-1BB (CD137) agonist

In some embodiments, the cell culture medium initiating the first amplification and/or rapid second amplification contains a TNFRSF agonist. In some embodiments, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule may contain any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The 4-1BB agonist or 4-1BB binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies); monoclonal antibodies (including full-length monoclonal antibodies); polyclonal antibodies; multispecific antibodies (e.g., bispecific antibodies); human, humanized, or chimeric antibodies; and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to 4-1BB, such as scFv molecules. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the 4-1BB agonist used in the methods and compositions disclosed in this invention includes anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectin, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. Agonistant anti-4-1BB antibodies are known to induce strong immune responses. (Lee et al., PLOSOne 2013, 8, e69677). In some embodiments, the 4-1BB agonist is an agonistant anti-4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In some embodiments, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utumumab, or urilumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

In some embodiments, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In some embodiments, polymeric 4-1BB agonists, such as trimeric or hexameric 4-1BB agonists (having three or six ligand-binding domains), can induce the formation of desirable receptor (4-1BBL) clusters and internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins containing three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic 4-1BB antibodies and fusion proteins induce strong immune responses. In some embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO:40) with high affinity and agonistic activity. In some embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO:40). In some embodiments, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO:41). The amino acid sequences of the 4-1BB antigens bound by the 4-1BB agonist or binding molecule are summarized in Table 5.

Table 5.4-1 shows the amino acid sequence of the BB antigen.

In some embodiments, the described compositions, processes, and methods include a 4-1BB agonist that binds to human or rodent 4-1BB at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at _{a KD} of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include 4-1BB agonists that _bind to human or rodent 4-1BB at a k-association rate of about 7.5 × ^{10⁵ 1} /M·s or faster, at a k _-association rate of about 7.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ 1/M·s or faster, at a k- _association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include binding to human or rodent 4-1BB at a k _{-dissociation} rate of about 2 × ^10⁻⁵ 1/s or _slower , at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about _2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 × ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/s or _slower , at about 2.6 × ^10⁻⁵ 1/s or _slower , or at about 2.7 × ^10⁻⁵ 1/s or slower. 4-1BB agonists that bind to human or rodent 4-1BB at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include a _4-1BB agonist that binds to human or rodent 4-1BB at an _IC50 of about ₁₀ nM or less, at about 9 nM or less, at about ₈ nM or less, at about 7 nM or less, at about ₆ nM or less, at about ₅ nM or less, at about ₄ nM or less, at about 3 nM or less, at about ₂ nM or less, or at about 1 nM or less _{. 50} combines with 4-1BB in humans or rodents.

In some embodiments, the 4-1BB agonist is utumumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant, or biosimilar thereof. Utumumab is available from Pfizer. Utumumab is an immunoglobulin G2-λ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of utumumab is illustrated in Table 6. Utumumab contains glycosylation sites at Asn59 and Asn292; intrachain disulfide bridges in the heavy chain at positions 22-96 ( _VH - _VL ), 143-199 ( _CH1 - _CL ), 256-316 ( _CH2 ), and 362-420 ( _CH3 ); and intrachain disulfide bridges at positions 22'-87' ( _VH - _VL ) and 136'-195' ( _CH1 -CL _). Intrachain disulfide bridges of the light chain; interchain heavy chain-heavy chain disulfide bridges located at IgG2A isoform positions 218-218, 219-219, 222-222 and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222 and 225-225 and at IgG2B isoform positions 219-130(2), 222-222 and 225-225; and interchain heavy chain-light chain disulfide bridges located at IgG2A isoform positions 130-213'(2), IgG2A/B isoform positions 218-213' and 130-213' and at IgG2B isoform positions 218-213'(2). The preparation and characterization of utumumab, as well as its variants and fragments, are described in U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678 and International Patent Application Publication No. WO 2012/032433 A1, the disclosures of which are incorporated herein by reference. The preclinical characteristics of utumumab are described in Fisher et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utumumab in various hematologic and solid tumor indications include US National Institutes of Health (NIH) clinicaltrials.gov identification numbers NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:42 and the light chain provided in SEQ ID NO:43. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utumumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:44, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:45, along with its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of utumumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity, such as 97%, 98%, 99%, or 100%, with an amino acid sequence that is 97%, 98%, 99%, or 100% sequence identity with a reference drug or reference biological product, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, which is utumumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, which is utumumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab.

Table 6. Amino acid sequences of 4-1BB agonist antibodies associated with utumumab.

In some embodiments, the 4-1BB agonist is the monoclonal antibody urogenumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biosimilar thereof. Ureogenumab is available from Bristol-Myers Squibb and Creative Biolabs. Ureogenumab is an immunoglobulin G4-κ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of urogenumab is illustrated in Table 7. Urelimab contains N-glycosylation sites at positions 298 (and 298”); intrachain disulfide bridges in the heavy chain at positions 22–95 ( _VH – _VL ), 148–204 ( _CH1 – _CL ), 262–322 ( _CH2 ), and 368–426 ( _CH3 ) (and at positions 22”–95”, 148”–204”, 262”–322”, and 368”–426”); and intrachain disulfide bridges in the heavy chain at positions 23”–88” ( _VH – _VL ) and 136”–196” ( _CH1 –CL _). (and intra-chain disulfide bridges at positions 23”'-88”' and 136”'-196”'); inter-chain heavy chain-heavy chain disulfide bridges at positions 227-227” and 230-230”; and inter-chain heavy chain-light chain disulfide bridges at positions 135-216”' and 135”-216”'. The preparation and characterization of urogenumab, as well as its variants and fragments, are described in U.S. Patent Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical characteristics of urogenumab are described in Segal et al., Clin. Cancer. Res. 2016, available at http://dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical trials of ureglucumab in various hematologic and solid tumor indications include NCT01775631, NCT02110082, NCT02253992, and NCT01471210 from the National Institutes of Health (NIH) clinicaltrials.gov.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:52 and the light chain provided in SEQ ID NO:53. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urinumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:54, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:55, and its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of urogenumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is uroreglumab.

Table 7. Amino acid sequences of 4-1BB agonist antibodies associated with urilumab.

In some embodiments, the 4-1BB agonist is selected from the group consisting of: 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), produced by a cell line registered as ATCC No. HB-11248 and disclosed in U.S. Patent No. 6,974,863. Antibodies disclosed in the following countries include: 5F4 (BioLegend 31 1503), C65-485 (BDPharmingen 559446), antibodies disclosed in US Patent Application Publication No. 2005/0095244, antibodies disclosed in US Patent No. 7,288,638 (e.g., 20H4.9-IgG1 (BMS-663031)), antibodies disclosed in US Patent No. 6,887,673 (e.g., 4E9 or BMS-554271), and antibodies disclosed in US Patent No. 7,214,493. Antibodies disclosed in U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, U.S. Patent No. 6,905,685 (e.g., 4E9 or BMS-554271), U.S. Patent No. 6,362,325 (e.g., 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), U.S. Patent No. 6,974,863 (e.g., 53A2), and U.S. Patent No. 6,210,669 (e.g., 5E9 or 5E2). The antibodies described in U.S. Patent No. 5,928,893 (1D8, 3B8, or 3E1), U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, and International Patent Application Publications Nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, as well as fragments, derivatives, conjugates, variants, or bioanalytes thereof, wherein the disclosure of each of the foregoing patents or patent applications is incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein described in the following: International Patent Application Publications No. WO 2008/025516 A1, WO 2009/007120A1, WO 2010/003766 A1, WO 2010/010051 A1 and WO 2010/078966 A1; U.S. Patent Application Publication No. US 201 The disclosures of U.S. Patent Nos. 1/0027218A1, 2015/0126709 A1, 2011/0111494A1, 2015/0110734 A1, and 2015/0126710 A1; and U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, are incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic fusion protein as depicted in structure IA (C-terminal Fc antibody fragment fusion protein) or structure IB (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof (see Figure 18). In structures IA and IB, the cylinder refers to an individual polypeptide-binding domain. Structures IA and IB contain three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL (4-1BB ligand, CD137 ligand (CD137L), or tumor necrosis factor superfamily member 9 (TNFSF9)) or antibodies binding to 4-1BB. These TNFRSF binding domains fold to form a trivalent protein, which is then linked to a second trivalent protein via an IgG1-Fc (including _CH3 and _CH2 domains). The IgG1-Fc then serves to link the two trivalent proteins together via disulfide bonds (elongated ellipses), thereby stabilizing the structure and providing an agonist capable of linking the intracellular signaling domains of six receptors to signaling proteins to form a signaling complex. A TNFRSF binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys. Any scFv domain design can be used, such as those described in: de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad et al., Clin. & Dev. Immunol. 2012, 980250; Monnier et al., Antibodies, 2013, 2, 193-208; or other references incorporated herein by reference. The structure of this form of fusion protein is described in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of structures I-A provided in Figure 18 are shown in Table 8. The Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO: 62), a complete hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). Preferred adapters for linking C-terminal Fc antibodies are selected from the embodiments provided in SEQ ID NO: 63 to SEQ ID NO: 72, including adapters suitable for fusing other polypeptides.

Table 8. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with C-terminal Fc-antibody fragment fusion protein design (structure I-A).

The amino acid sequences of the other polypeptide domains of structures I-B provided in Figure 18 are shown in Table 9. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SED ID NO:74 to SEQ ID NO:76.

Table 9. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with N-terminal Fc-antibody fragment fusion protein design (structures I-B).

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B includes one or more 4-1BB binding domains selected from the group consisting of: variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains selected from the variable heavy and variable light chains described in Table 10, any combination of the aforementioned variable heavy and variable light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:77. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a soluble 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:78.

In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:43 and SEQ ID NO:44, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the _VH and _VL sequences provided in Table 10, wherein the _VH and _VL domains are connected by linkers.

Table 10. Applicable to the 4-1BB binding domain in fusion proteins or other polypeptide domains applicable to scFv 4-1BB agonist antibodies.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stem region (which promotes trimerization and provides a distance from the cell membrane, but is not part of the 4-1BB binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some embodiments, the 4-1BB agonist is a BPS Bioscience 4-1BB agonist antibody, catalog number 79097-2, available from BPS Bioscience in San Diego, California, USA. In other embodiments, the 4-1BB agonist is a Creative Biolabs 4-1BB agonist antibody, catalog number MOM-18179, available from Creative Biolabs in Shirley, NY, USA.

3. OX40 (CD134) agonist

In some embodiments, the TNFRSF agonist is an OX40 (CD134) agonist. The OX40 agonist can be any OX40-binding molecule known in the art. The OX40-binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX40. The OX40 agonist or OX40-binding molecule may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The OX40 agonist or OX40-binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized or chimeric antibodies, and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to OX40, such as scFv molecules. In some embodiments, the OX40 agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the OX40 agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the OX40 agonist used in the methods and compositions of this disclosure includes anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 adenettin, anti-OX40 domain antibodies, single-chain anti-OX40 fragments, heavy-chain anti-OX40 fragments, light-chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. In some embodiments, the OX40 agonist is an agonistic anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

In some embodiments, the OX40 agonist or OX40 binding molecule may also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun et al., J. Immunother. 2009, 182, 1481-89. In some embodiments, poly-OX40 agonists, such as trimeric or hexameric OX40 agonists (having three or six ligand-binding domains), can induce clustering of the preferred receptor (OX40L) and the formation of internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic OX40 antibodies and fusion proteins can induce strong immune responses. (Curti et al., Cancer Res. 2013, 73, 7189-98). In some embodiments, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficiently reducing toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC), such as NK cell toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the OX40 agonist is characterized by binding to human OX40 (SEQ ID NO: 85) with high affinity and agonistic activity. In some embodiments, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 85). In some embodiments, the OX40 agonist is a binding molecule that binds to mouse OX40 (SEQ ID NO: 86). Table 11 summarizes the amino acid sequences of the OX40 antigen bound to the OX40 agonist or binding molecule.

Table 11. Amino acid sequence of OX40 antigen.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind human or rodent OX40 at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at a _KD of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that _bind to human or rodent OX40 at a k-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at a k _{-dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about 2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 _× ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/ _s or slower, and at about 2.6 × ^10⁻⁵ 1/s or slower. It binds to human or rodent OX40 at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.7 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at an _IC50 of about 10 nM or less, at an IC50 of about 9 nM or less, at an _IC50 of about 8 nM or less _, at an _IC50 of about 7 nM or less, at an IC50 of about 6 nM or less, at an _IC50 of about ₅ nM or less, at an IC50 of about 4 nM or less, at an _IC50 of about 3 nM or less, at _{an IC50} of about 2 nM or less, or at an _IC50 of about 1 nM or less.

In some implementations, the OX40 agonist is tavoricumab, also known as MEDI 0562 or MEDI-0562. Tavoricumab is available from MedImmune, a subsidiary of AstraZeneca. Tavoricumab is a humanized and chimeric monoclonal antibody against immunoglobulin G1-κ [Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)]. The amino acid sequence of tavoricumab is illustrated in Table 12. Tavocitumab contains N-glycosylation sites at positions 301 and 301", with fucosylated complex biantennary CHO-type glycans; intrachain disulfide bridges in the heavy chain at positions 22-95 ( _VH - _VL ), 148-204 ( _CH1 - _CL ), 265-325 ( _CH2 ), and 371-429 ( _CH3 ) (and at positions 22"-95", 148"-204", 265"-325" and 371"-429"); and intrachain disulfide bridges in the heavy chain at positions 23"-88" ( _VH - _VL ) and 134"-194" ( _CH1 -CL ₎ . Intra-chain disulfide bridges of the light chain at positions 23”'-88”' and 134”'-194”'; inter-chain heavy chain-heavy chain disulfide bridges at positions 230-230” and 233-233”'; and inter-chain heavy chain-light chain disulfide bridges at positions 224-214”' and 224”-214”'. Current clinical trials of tavoricumab in various solid tumor indications include NCT02318394 and NCT02705482 from the National Institutes of Health clinicaltrials.gov.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:87 and the light chain provided in SEQ ID NO:88. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of tavoricumab. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:89, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:90, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90 _, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises an scFv antibody, said scFv antibody comprising _VH and _VL regions _, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of the OX40 agonist approved by a regulatory agency, referencing tavoricumab. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab.

Table 12. Amino acid sequences of OX40 agonist antibodies associated with tavoricumab.

In some embodiments, the OX40 agonist is 11D4, a fully human antibody available from Pfizer. The preparation and characterization of 11D4 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is set forth in Table 13.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:97 and the light chain provided in SEQ ID NO:98. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:99, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:100, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100 _, respectively. In some embodiments, the OX40 agonist comprises _VH and VL _regions , each at least 97% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody approved by a regulatory agency with reference to 11D4. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug product or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 11D4.

Table 13. Amino acid sequences of OX40 agonist antibodies associated with 11D4.

In some embodiments, the OX40 agonist is 18D8, a fully human antibody available from Pfizer. The preparation and characterization of 18D8 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is set forth in Table 14.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:107 and the light chain provided in SEQ ID NO:108. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively.

In some embodiments, the OX40 agonist comprises a heavy chain and a light chain CDR or variable region (VR) of 18D8. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:109, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:110, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO: ₁₁₀ , respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to 18D8. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence having at least 97%, such as 97%, 98%, 99%, or 100%, of a reference drug product or reference biological product, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 18D8.

Table 14. Amino acid sequences of OX40 agonist antibodies associated with 18D8.

In some embodiments, the OX40 agonist is Hu119-122, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu119-122 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is set forth in Table 15.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:117, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:118 and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu119-122. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122.

Table 15. Amino acid sequences of OX40 agonist antibodies associated with Hu119-122.

In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu106-222 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is set forth in Table 16.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:125, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:126, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 96% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu106-222. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222.

Table 16. Amino acid sequences of OX40 agonist antibodies associated with Hu106-222.

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B12). MEDI6469 is a murine monoclonal antibody. (Weinberg et al., J. Immunother. 2006, 29, 575-585). In some embodiments, the OX40 agonist is an antibody produced from a 9B12 hybridoma and hosted by Biovest Corporation (Malvern, MA, USA), as described in Weinberg et al., J. Immunother. 2006, 29, 575-585, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the antibody comprises the CDR sequence of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of MEDI6469.

In some embodiments, the OX40 agonist is L106 BD (Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the CDR of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the CDR of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some implementations, the OX40 agonist is a murine monoclonal antibody anti-mCD134/mOX40 (clone OX86), available from BioXcell's InVivoMAb in West Lebanon, New Hampshire.

In some embodiments, the OX40 agonist is selected from the OX40 agonists described below: International Patent Application Publications WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191 and WO 2014/148895; European Patent Application EP 0672141; US Patent Application Publication US2010/136030, ... US Nos. 2014/377284, 2015/190506, and 2015/132288 (including clones 20E5 and 12H3); and US U.S. Patents 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the OX40 agonist is an OX40 agonistic fusion protein as depicted in structures I-A (C-terminal Fc-antibody fragment fusion protein) or I-B (N-terminal Fc-antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof. The characteristics of structures I-A and I-B have been described above and in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequences of the polypeptide domains of structure I-A provided in Figure 18 are shown in Table 9. The Fc domain preferably comprises an intact constant domain (amino acids 17-230 of SEQ ID NO: 62), an intact hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). The preferred adapter for linking the C-terminal Fc antibody can be selected from the embodiments provided in SEQ ID NO:63 to SEQ ID NO:72, including adapters suitable for fusing other peptides. Similarly, the amino acid sequences of the polypeptide domains of structures I-B provided in Figure 18 are shown in Table 10. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SEQ ID NO:74 to SEQ ID NO:76.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40 binding domains selected from the group consisting of: the variable heavy and light chains of tavocisumab, the variable heavy and light chains of 11D4, the variable heavy and light chains of 18D8, the variable heavy and light chains of Hu119-122, the variable heavy and light chains of Hu106-222, the variable heavy and light chains selected from the variable heavy and light chains described in Table 17, any combination of the aforementioned variable heavy and light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 133. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the soluble OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 134. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 135.

In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:127 and SEQ ID NO:128, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO: 125 and SEQ ID NO: 126, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and VL regions, respectively, that are at least 95% identical to the _VH and _VL sequences provided in Table 17, wherein the _VH and _{VL regions} are connected by linkers.

Table 17. OX40 binding domains suitable for use as fusion proteins (e.g., structures I-A and I-B) or other polypeptide domains suitable for use as scFv OX40 agonist antibodies.

In some embodiments, the OX40 agonist is an OX40-agonistic single-chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain further comprising an additional domain at an N-terminus and/or a C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble OX40-binding domain; (ii) a first peptide linker; (iii) a second soluble OX40-binding domain; (iv) a second peptide linker; and (v) a third soluble OX40-binding domain further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domains are Fab or Fc fragment domains, wherein each of the soluble OX40-binding domains lacks a stem region (which facilitates trimerization and provides a distance from the cell membrane, but is not part of the OX40-binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain; (ii) a first peptide linker; (iii) a second soluble TNF superfamily cytokine domain; (iv) a second peptide linker; and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily cytokine domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonist fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In some embodiments, the OX40 agonist is an OX40-agonistic scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some implementations, the OX40 agonist is Creative Biolabs' OX40 agonist monoclonal antibody MOM-18455, which is available from Creative Biolabs in Sherry, New York, USA.

In some implementations, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, which is available from BioLegend, Inc., San Diego, California, USA.

B. Optional cell viability analysis

Optionally, after initiating the first expansion (sometimes referred to as initial bulk expansion), cell viability can be determined using standard assays known in the art. Therefore, in some embodiments, the method includes performing cell viability determination after initiating the first expansion. For example, a trypan blue exclusion assay can be performed on the bulk TIL sample, which selectively labels dead cells and allows for viability assessment. Other assays used to test viability may include (but are not limited to) an alamar blue assay and an MTT assay.

1. Cell counting, viability, and flow cytometry

In some embodiments, cell count and/or viability are measured. Expression of biomarkers (e.g., but not limited to CD3, CD4, CD8, and CD56, and any other biomarkers disclosed or described herein) can be measured by flow cytometry using a FACSCanto ^™ flow cytometer (BD Biosciences), with antibodies, such as (but not limited to) those available from BD Biosciences (BD Biosciences, San Jose, CA). Cells can be manually counted using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art (including, but not limited to, trypan blue staining). Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0282694, which is incorporated herein by reference in its entirety. Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0280436 or International Patent Application Publication No. WO/2018/081473, both of which are incorporated herein by reference in their entirety for all purposes.

In some cases, the primary TIL population can be immediately cryopreserved using the methods discussed below. Alternatively, the primary TIL population can undergo re-expansion (REP) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs will be used in therapy, the primary or REP TIL population can be genetically modified for appropriate treatment.

2. Cell Culture

In some embodiments, the method for amplifying TILs (including those discussed above and illustrated in Figures 1 and 8, particularly those exemplified in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may include the use of about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the culture medium is serum-free. In some embodiments, the culture medium used to initiate the first amplification is serum-free. In some embodiments, the culture medium used to initiate the second amplification is serum-free. In some embodiments, the culture medium used to initiate both the first and second amplifications (also referred to as rapid second amplification) is serum-free. In some embodiments, TIL number amplification uses no more than one type of cell culture medium. Any suitable cell culture medium can be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, CA). In this regard, the method of the present invention advantageously reduces the amount and number of culture media types required for TIL number amplification. In some embodiments, TIL number amplification may include feeding cells at a frequency not exceeding once every three or four days. Amplifying cell numbers in an aerated container simplifies the procedure required for cell number amplification by reducing the feeding frequency needed.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME)-free.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for a period of about 1 to 8 days, for example, about 7 days as the initiation of the first amplification, or about 8 days as the initiation of the first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for a period of about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for about 1 to 7 days (e.g., about 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TILs to a second ventilated container and amplifying the number of TILs in the second ventilated container for about 7 to 14 days or about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days, about 10 days, or about 11 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for approximately 1 to 7 days (e.g., approximately 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium comprising IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for approximately 7 to 11 days (e.g., approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, or approximately 11 days), the second ventilated container containing a cell culture medium comprising IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, TILs are amplified in a breathable container. Breathable containers have been used to amplify TILs, using PBMCs, and employing methods, compositions, and apparatus known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In some embodiments, TILs are amplified in a breathable bag. In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the Xuri Cell Expansion System W25 (GE Healthcare)). In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the WAVE bioreactor system, also known as the Xuri Cell Expansion System W5 (GE Healthcare)). In some embodiments, the cell expansion system includes a breathable cell bag with a volume selected from the group consisting of: about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

In some embodiments, TILs can be expanded in G-REX culture flasks (available from Wilson Wolf Manufacturing). Such embodiments expand the cell population from approximately 5 × ^10⁵ cells/cm² to 10 × ^10⁶ to 30 × ^10⁶ cells/cm². In some embodiments, this is not performed feedering. In some embodiments, this is not performed feedering, as long as the culture medium in the G-REX culture flask is at a height of approximately 10 cm. In some embodiments, this is not performed feedering, but one or more cytokines are added. In some embodiments, the cytokines are added as a bolus, without needing to be mixed with the culture medium. Such containers, apparatuses, and methods are known in the art and have been used to amplify TILs, and include those described in the following: U.S. Patent Application Publication No. US2014/0377739A1, International Publication No. WO 2014/210036A1, U.S. Patent Application Publication No. US 2013/0115617A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Patent No. US 8,809,050 B2, International Publication No. WO 2011/072088A2, U.S. Patent Application Publication No. US2016/0208216A1, U.S. Patent Application Publication No. US2012/0244133 A1, International Publication No. WO 2012/129201A1, and U.S. Patent Application Publication No. US2013/0102075. The disclosures of U.S. Patent No. A1, U.S. Patent No. 8,956,860 B2, International Publication No. WO 2013/173835A1, and U.S. Patent Application Publication No. US2015/0175966 A1 are incorporated herein by reference. Similar processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

Optional gene knockdown or gene knockout in C.TIL

In some embodiments, the amplified TILs of the present invention are further manipulated before, during, or after the amplification step, including during a closed, aseptic manufacturing process (each as provided herein), to temporarily alter protein expression. In some embodiments, the temporarily altered protein expression is due to temporary gene editing. In some embodiments, the amplified TILs of the present invention are treated with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in TILs. In some embodiments, TFs and/or other molecules capable of temporarily altering protein expression provide altered tumor antigen expression and/or altered number of tumor antigen-specific T cells in the TIL population.

In some embodiments, the method includes gene editing of a TIL population. In some embodiments, the method includes gene editing of a first TIL population, a second TIL population, and/or a third TIL population.

In some embodiments, the invention includes gene editing via nucleotide insertion, such as via ribonucleic acid (RNA) insertion, including the insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA) into a TIL population to promote or inhibit the expression of one or more proteins, as well as a combination of promoting one group of proteins and inhibiting another group of proteins.

In some embodiments, the amplified TILs of the present invention undergo a temporary alteration of protein expression. In some embodiments, the temporary alteration of protein expression occurs in the host TIL population prior to the first amplification, including, for example, the TIL population obtained from step A as indicated in, for example, Figure 8 (particularly Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs during the first amplification, including, for example, the TIL population obtained from step B as indicated in Figure 8 (e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs after the first amplification, including, for example, the TIL population transitioning between the first and second amplifications (e.g., the second TIL population described herein), the TIL population obtained from, for example, step B as indicated in Figure 8, and included in step C. In some embodiments, the temporary alteration of protein expression occurs in the main TIL population prior to the second amplification, including, for example, the TIL population obtained from, for example, step C as indicated in Figure 8, and prior to its amplification in step D. In some embodiments, the temporary alteration of protein expression occurs during a second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8 (e.g., a third TIL population). In some embodiments, the temporary alteration of protein expression occurs after the second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8.

In some embodiments, the method for temporarily altering protein expression in a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. In some embodiments, the method for temporarily altering protein expression in a TIL population includes a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include transfection steps using methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

In some embodiments, temporary alteration of protein expression induces an increase in stem memory T cells (TSCMs). TSCMs are early progenitor cells of antigen-experiencing central memory T cells. TSCMs typically exhibit the long-term survival, self-renewal, and pluripotent capabilities that define stem cells and are generally required for the production of effective TIL products. In mouse models of adoptive cell transfer, enhanced antitumor activity of TSCMs compared to other subsets of T cells has been demonstrated. In some embodiments, temporary alteration of protein expression induces a TIL population with a composition containing a high proportion of TSCMs. In some embodiments, temporary alteration of protein expression induces an increase in the percentage of TSCMs of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, a temporary alteration of protein expression causes a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCM in a TIL population. In some embodiments, a temporary alteration of protein expression causes a TIL population with at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM. In some implementations, a temporary alteration in protein expression results in a therapeutic TIL population having at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM.

In some implementations, a temporary alteration of protein expression induces antigen-experiencing T-cell rejuvenation. In some implementations, rejuvenation includes, for example, increased proliferation, increased T-cell activation, and/or increased antigen recognition.

In some implementations, temporarily altering protein expression changes the expression of a large portion of T cells to preserve the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression does not alter the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression maintains the tumor-derived TCR reservoir.

In some implementations, temporary alteration of a protein induces a change in the expression of a specific gene. In some implementations, the temporary alteration of protein expression targets genes including (but not limited to) the following: PD-1 (also known as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, chimeric co-stimulatory receptor (CCR), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, and CC. R5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some implementations, protein expression targeting genes selected from the following groups is temporarily altered: PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL. 2(MCP-1), CCL3(MIP-1α), CCL4(MIP1-β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some embodiments, protein expression is temporarily altered to target PD-1. In some embodiments, protein expression is temporarily altered to target TGFBR2. In some embodiments, protein expression is temporarily altered to target CCR4/5. In some embodiments, protein expression is temporarily altered to target CBL-B. In some embodiments, protein expression is temporarily altered to target CISH. In some embodiments, protein expression is temporarily altered to target CCR (chimeric co-stimulatory receptor). In some embodiments, protein expression is temporarily altered to target IL-2. In some embodiments, protein expression is temporarily altered to target IL-12. In some embodiments, protein expression targeting IL-15 is temporarily altered. In some embodiments, protein expression targeting IL-21 is temporarily altered. In some embodiments, protein expression targeting NOTCH 1/2 ICD is temporarily altered. In some embodiments, protein expression targeting TIM3 is temporarily altered. In some embodiments, protein expression targeting LAG3 is temporarily altered. In some embodiments, protein expression targeting TIGIT is temporarily altered. In some embodiments, protein expression targeting TGFβ is temporarily altered. In some embodiments, protein expression targeting CCR1 is temporarily altered. In some embodiments, protein expression targeting CCR2 is temporarily altered. In some embodiments, protein expression targeting CCR4 is temporarily altered. In some embodiments, protein expression targeting CCR5 is temporarily altered. In some embodiments, protein expression targeting CXCR1 is temporarily altered. In some embodiments, protein expression targeting CXCR2 is temporarily altered. In some embodiments, protein expression targeting CSCR3 is temporarily altered. In some embodiments, protein expression targeting CCL2 (MCP-1) is temporarily altered. In some embodiments, protein expression targeting CCL3 (MIP-1α) is temporarily altered. In some embodiments, protein expression targeting CCL4 (MIP1-β) is temporarily altered. In some embodiments, protein expression targeting CCL5 (RANTES) is temporarily altered. In some embodiments, protein expression targeting CXCL1 is temporarily altered. In some embodiments, protein expression targeting CXCL8 is temporarily altered. In some embodiments, protein expression targeting CCL22 is temporarily altered. In some embodiments, protein expression targeting CCL17 is temporarily altered. In some embodiments, protein expression targeting VHL is temporarily altered. In some embodiments, protein expression targeting CD44 is temporarily altered. In some embodiments, protein expression targeting PIK3CD is temporarily altered. In some embodiments, protein expression targeting SOCS1 is temporarily altered. In some embodiments, protein expression targeting thymocyte selection-associated high migration group (HMG) box (TOX) is temporarily altered. In some embodiments, protein expression targeting ankyrin repeat domain 11 (ANKRD11) is temporarily altered. In some embodiments, protein expression targeting BCL6 co-inhibitor (BCOR) is temporarily altered. In some embodiments, protein expression targeting cAMP protein kinase A (PKA) is temporarily altered.

In some embodiments, a temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors. In some embodiments, the chemokine receptors overexpressed due to temporary protein expression include receptors with ligands, said ligands including (but not limited to) CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.

In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and/or TGFβ (including resulting in, for example, TGFβ pathway blockade). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CBLB (CBL-B). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CISH. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of TIGIT. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors to, for example, improve TIL transport or motility to tumor sites. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CCRs (chimeric co-stimulatory receptors). In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors selected from the group consisting of: CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.

In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins. In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins selected from the group consisting of IL-2, IL-12, IL-15, and/or IL-21.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of NOTCH 1/2 ICD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of VHL. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CD44. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of PIK3CD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of SOCS1.

In some implementations, temporarily altering protein expression causes a decrease and/or reduction in the expression of cAMP protein kinase A (PKA).

In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of two molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one molecule selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of the following: PD-1, CTLA-4, LAG-3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one of the following: CTLA-4, LAG3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CTLA-4. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIM-3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and PD-1. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and TIGIT.

In some implementations, adhesion molecules selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof are inserted into a first TIL population, a second TIL population, or a collected TIL population via gamma retrovirus or lentivirus methods (e.g., increased expression of adhesion molecules).

In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBL-B, and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, expression is reduced by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some implementations, expression is reduced by at least approximately 90%. In some implementations, expression is reduced by at least approximately 95%. In some implementations, expression is reduced by at least approximately 99%.

In some embodiments, expression is increased by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some implementations, expression increases by at least about 90%. In some implementations, expression increases by at least about 95%. In some implementations, expression increases by at least about 99%.

In some embodiments, the temporary alteration of protein expression is induced by treating the TIL with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in the TIL. In some embodiments, intracellular delivery of transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression is performed using a microfluidic platform without SQZ vectors. Such methods have demonstrated the ability to deliver proteins comprising transcription factors to a variety of primary human cell types, including T cells, as described in U.S. Patent Application Publications Nos. US2019/0093073A1, US2018/0201889A1, and US2019/0017072A1, the disclosures of which are each incorporated herein by reference. Such methods can be used in this invention to expose a TIL population to transcription factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression provide an increase in the expression of tumor antigens and/or the number of tumor antigen-specific T cells in the TIL population, thereby leading to TIL population reprogramming and increased therapeutic efficacy of the reprogrammed TIL population compared to the non-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in effector T cells and/or central memory T cell subsets relative to the initial or previous TIL population (i.e., prior to reprogramming), as described herein.

In some embodiments, the transcription factor (TF) includes (but is not limited to) TCF-1, NOTCH 1/2 ICD, and/or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1/2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered with an induced pluripotent stem cell culture (iPSC), such as a commercially available KNOCKOUT serum alternative (Gibco/ThermoFisher), to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered with an iPSC mixture to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is not administered with an iPSC mixture. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some implementations, reprogramming causes a percentage increase in TSCM of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% of TSCM.

In some embodiments, the method of temporarily altering protein expression as described above may be combined with a method of genetically modifying TIL populations, including a step of stabilizing the incorporation of genes for producing one or more proteins. In some embodiments, the method includes a step of genetically modifying TIL populations. In some embodiments, the method includes genetically modifying a first TIL population, a second TIL population, and/or a third TIL population. In some embodiments, the method of genetically modifying TIL populations includes a retroviral transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in: Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71; and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a gamma-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include transposases provided as DNA expression vectors or as expressible RNA or proteins, such that long-term expression of the transposase does not occur in transgenic cells, for example, transposases provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems, including salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, are described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In some implementations, temporary alteration of protein expression in the TIL is induced by small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, which is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where it interferes with the expression of specific genes having complementary nucleotide sequences.

In some implementations, the temporary alteration of protein expression is a reduction in expression. In some implementations, the temporary alteration of protein expression in the TIL is induced by self-delivered RNA interference (sdRNA), which is a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or _-OCH3 ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand bound to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence. sdRNA is a covalently and hydrophobically modified RNAi compound that does not require a delivery medium to enter the cell. sdRNA is typically an asymmetrically modified nucleic acid molecule with a very small double-stranded region. sdRNA molecules typically contain single-stranded and double-stranded regions, and various chemical modifications can be incorporated into these regions. Furthermore, as described herein, sdRNA molecules can be linked to hydrophobic conjugates such as conventional and higher sterol molecules. sdRNAs and related methods for their preparation have been extensively described, for example, in U.S. Patent Application Publications US2016/0304873 A1, US2019/0211337A1, US2009/0131360A1, and US2019/0048341A1, and U.S. Patents 10,633,654 and 10,913,948B2, the disclosures of which are each incorporated herein by reference. To optimize sdRNA structure, chemical properties, target location, sequence preference, etc., an algorithm has been developed and used for sdRNA efficacy prediction. Based on these analyses, a functional sdRNA sequence is generally defined as one whose expression is reduced by more than 70% at a concentration of 1 μM, with a probability exceeding 40%.

Double-stranded DNA (dsRNA) can generally be used to define any molecule containing a pair of complementary RNA strands, typically sense (passenger) and antisense (guide) strands, and may include single-stranded overhang regions. Unlike siRNA, the term dsRNA generally refers to a precursor molecule comprising the sequence of an siRNA molecule, which is released from a larger dsRNA molecule by the action of a lysin system (including Dicer).

In some implementations, the method includes temporarily altering protein expression in a population of TILs (including TILs modified to express CCR), including using self-delivered RNA interference (sdRNA), which is, for example, a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or -OCH ₃ ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand that binds to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Methods using siRNA and sdRNA have been described in the following: Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248; Byrne et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93, the disclosures of which are incorporated herein by reference. In some embodiments, siRNA delivery is accomplished using electroporation or cell membrane disruption (e.g., squeezing or SQZ). In some embodiments, delivery of siRNA or sdRNA to a TIL population does not require electroporation, SQZ, or other methods; the TIL population is actually exposed to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, the method includes delivery of siRNA or sdRNA to a TIL population, which involves exposing the TIL population to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 10 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration between 0.1 μM/10,000 TILs and 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days, wherein the exposure to siRNA or sdRNA is performed two, three, four, or five times by adding fresh siRNA or sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. Patent Application Publications Nos. US 2011/0039914A1, US 2013/0131141A1 and US 2013/0131142A1, and U.S. Patent No. 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA is inserted into the TIL population during manufacturing. In some embodiments, the sdRNA encodes RNA that interferes with the following: NOTCH 1/2 ICD, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and/or CBL-B. In some embodiments, the expression reduction is determined based on the percentage of gene silencing assessed, for example, by flow cytometry and/or qPCR. In some embodiments, the expression reduction is approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

Self-deliverable RNAi technology based on chemically modified siRNA can be used in the methods of this invention to successfully deliver sdRNA to TILs as described herein. The combination of backbone modification with asymmetric siRNA structures and hydrophobic ligands (see, for example, Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93 and U.S. Patent Application Publication No. 2016/0304873A1, the disclosure of which is incorporated herein by reference) allows sdRNA to permeate cultured mammalian cells simply by adding it to the culture medium, utilizing the nuclease stability of sdRNA without the need for additional formulations and methods. This stability allows for the support of constant levels of RNAi-mediated reduction in target gene activity simply by maintaining an effective concentration of sdRNA in the culture medium. Although not bound by theory, backbone stabilization of sdRNA provides extended reductions in gene expression that can persist for months in non-dividing cells.

In some embodiments, over 95% TIL transfection efficiency and target expression reduction occur through various specific siRNAs or sdRNAs. In some embodiments, siRNAs or sdRNAs containing several unmodified ribose residues undergo fully modified sequence substitutions to increase the potency and/or lifetime of the RNAi effect. In some embodiments, the expression reduction effect lasts for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or longer. In some embodiments, the expression reduction effect decreases after siRNA or sdRNA treatment of TILs for 10 days or longer. In some embodiments, target expression is reduced by more than 70%. In some embodiments, target expression in TILs is reduced by more than 70%. In some embodiments, expression reduction in the PD-1/PD-L1 pathway allows TILs to exhibit a more potent in vivo effect, which in some embodiments is due to avoiding the inhibitory effects of the PD-1/PD-L1 pathway. In some embodiments, TIL proliferation is increased due to reduced PD-1 expression by siRNA or sdRNA.

In some embodiments, the sdRNA sequence used in this invention exhibits a 70% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 75% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 80% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 85% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 90% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 95% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 99% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM to about 4 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 4.0 μM.

In some embodiments, the siRNA or sdRNA oligonucleotide agent comprises one or more modifications to increase the stability and/or efficacy of the therapeutic agent and to enable efficient delivery of the oligonucleotide to the cells or tissues to be treated. Such modifications may include 2'-O-methyl modifications, 2'-O-fluorine modifications, dithiophosphate modifications, 2'F-modified nucleotides, 2'-O-methyl-modified and/or 2'-deoxynucleotides. In some embodiments, the oligonucleotide is modified to include one or more hydrophobic modifications, including, for example, sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and/or phenyl. In some embodiments, the chemically modified nucleotide is a combination of thiophosphate, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and thiophosphate. In some embodiments, the sugar may be modified, and the modified sugar may include (but is not limited to) D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halogen (including 2'-fluorine), T-methoxyethoxy, 2'-allyloxy ( _-OCH₂CH = _CH₂ ), 2'-propynyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano. In some embodiments, the sugar moiety may be a hexose and incorporated into an oligonucleotide, such as Augustyns et al., Nucl. Acids. Res. 1992, 18, 4711, the disclosure of which is incorporated herein by reference.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is double-stranded along its entire length, i.e., without a dangling single-stranded sequence at either end of the molecule, i.e., a blunt end. In some embodiments, individual nucleic acid molecules may have different lengths. In other words, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is not double-stranded along its entire length. For example, when using two separate nucleic acid molecules, one of the molecules, such as the first molecule containing an antisense sequence, may be longer than the second molecule with which it hybridizes (leaving a portion of the molecule single-stranded). In some embodiments, when using a single nucleic acid molecule, a portion of the molecule at either end may remain single-stranded.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention contain mismatches and/or loops or protrusions, but are double-stranded for at least about 70% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded for at least about 80% of the oligonucleotide length. In other embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 90%-95% of the oligonucleotide length. In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 96%-98% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention contain at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

In some embodiments, siRNA or sdRNA oligonucleotides may be substantially protected against nucleases, for example, by modifying the 3' or 5' bonds, as described in U.S. Patent No. 5,849,902, the disclosure of which is incorporated herein by reference. For example, oligonucleotides may be made resistant by incorporating a “blocking group.” As used herein, the term “blocking group” refers to a substituent (e.g., other than an OH group) that can be linked to the oligonucleotide or nucleoside as a protecting or coupling group for synthesis (e.g., FITC, propyl ( _CH₂ - _CH₂ - _CH₃ ), diol (-O- _CH₂ - ^CH₂ _- O-) phosphate ( _PO₃²⁻ ), hydrogen phosphonate, or aminophosphite). A “blocking group” may also include a “terminal blocking group” or “exonuclease blocking group” that protects the 5' and 3' ends of the oligonucleotide, comprising modified nucleotide and non-nucleotide exonuclease resistant structures.

In some implementations, at least a portion of the continuous polynucleotides within the siRNA or sdRNA are linked by substituents, such as phosphate thioesters.

In some embodiments, chemical modification may result in at least 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 13% Cellular uptake of siRNA or sdRNA is enhanced at 0%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, multiple C and U residues contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U may contain hydrophobic modifications. In some embodiments, all C and U contain hydrophobic modifications.

In some embodiments, siRNA or sdRNA molecules exhibit enhanced endosome release via the incorporation of a protonable amine. In some embodiments, the protonable amine is incorporated into the sense strand (the molecular portion discarded after RISC loading). In some embodiments, the siRNA or sdRNA compounds of the present invention comprise an asymmetric compound comprising a double-stranded region (required for efficient RISC entry, 10-15 bases long) and a single-stranded region 4-12 nucleotides long; a double-stranded region having 13 nucleotides. In some embodiments, a single-stranded region of 6 nucleotides is employed. In some embodiments, the single-stranded region of the siRNA or sdRNA comprises 2-12 phosphate thioester nucleotide links (referred to as phosphate thioester modification). In some embodiments, 6-8 phosphate thioester nucleotide links are employed. In some embodiments, the siRNA or sdRNA compounds of the present invention further include unique chemical modification patterns that provide stability and compatibility with RISC entry. For example, the guide strand can also be modified by any chemical modification that demonstrates stability without interfering with RISC entry. In some implementations, the chemical modification pattern in the guide chain includes 2'F modification of most C and U nucleotides and phosphorylation at the 5' end.

In some embodiments, at least 30% of the nucleotides in the siRNA or sdRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6 3%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides are modified. In some embodiments, 100% of the nucleotides in the siRNA or sdRNA are modified.

In some embodiments, the siRNA or sdRNA molecule has a very small double-stranded region. In some embodiments, the double-stranded region of the molecule is between 8 and 15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides in length. There may be 100% complementarity between the guide strand and the passenger strand, or one or more mismatches may exist between the guide strand and the passenger strand. In some embodiments, the molecule has a blunt end or a dangling structure with one nucleotide at one end. The single-stranded region of the molecule is between 4 and 12 nucleotides in length in some embodiments. In some embodiments, the single-stranded region may be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In some embodiments, the single-stranded region may also be less than 4 nucleotides or greater than 12 nucleotides in length. In some embodiments, the single-stranded region is 6 or 7 nucleotides in length.

In some embodiments, the siRNA or sdRNA molecules exhibit increased stability. In some cases, the chemically modified siRNA or sdRNA molecules have a half-life in culture medium longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours, including any intermediate values. In some embodiments, the siRNA or sdRNA has a half-life in culture medium exceeding 12 hours.

In some embodiments, siRNA or sdRNA is optimized to increase efficacy and/or reduce toxicity. In some embodiments, the nucleotide length of the guide strand and/or passenger strand and/or the number of phosphate thioester modifications in the guide strand and/or passenger strand can affect the efficacy of the RNA molecule in some respects, while replacing 2'-fluorine (2'F) modifications with 2'-O-methyl (2'OMe) modifications can affect the toxicity of the molecule in some respects. In some embodiments, reducing the 2'F content of the molecule is expected to reduce the toxicity of the molecule. In some embodiments, the number of phosphate thioester modifications in the RNA molecule can affect the efficiency of molecule uptake into cells, for example, passive uptake of molecules into cells. In some embodiments, siRNA or sdRNA does not have 2'F modifications, but is characterized by equivalent efficacy in cellular uptake and tissue penetration.

In some embodiments, the guide chain is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate ester modifications. For example, the guide chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate ester-modified nucleotides. The guide chain may contain one or more modifications that confer increased stability without interfering with RISC entry. Phosphate-ester-modified nucleotides, such as thiophosphate-modified nucleotides, may be at the 3' end, 5' end, or throughout the entire guide chain. In some embodiments, the 3' end 10 nucleotides of the guide chain contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 thiophosphate-modified nucleotides. The guide chain may also contain 2'F and/or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at position 1 in the guide chain (the nucleotide at the 5' end of the guide chain) is modified with 2'OMe and/or phosphorylated. C and U nucleotides within the guide chain may be modified with 2'F. For example, the C and U nucleotides at positions 2-10 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'F. The C and U nucleotides within the guide chain may also be modified with 2'OMe. For example, the C and U nucleotides at positions 11-18 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'OMe. In some embodiments, the nucleotides at the 3' end of the guide chain are unmodified. In some embodiments, most of the C and U nucleotides within the guide chain are modified with 2'F, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, the 5' end of the guide chain is phosphorylated, and the C or U at positions 2-10 are modified with 2'F.

Self-deliverable RNAi technology provides a method for directly transfecting cells with an RNAi agent (whether siRNA, sdRNA, or other RNAi agents) without additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity, and ease of use are characteristics of the compositions and methods, which offer significant functional advantages over conventional siRNA-based techniques, and therefore the sdRNA method is employed in several embodiments of the method for reducing target gene expression in TILs of the present invention. The sdRNA method allows for the direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues in vitro and in vivo. The sdRNAs described in some embodiments of the invention herein are available from Advirna LLC, Worcester, MA, USA.

siRNA and sdRNA can be formed by hydrophobically modified siRNA-antisense oligonucleotide hybridization and are disclosed, for example, in Byrne et al., J. Ocular Pharmacol. Therapeut., 2013, 29, 855-864, the contents of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA oligonucleotides may be delivered to the TILs described herein using sterile electroporation. In some embodiments, the method includes sterile electroporation of TIL populations to deliver siRNA or sdRNA oligonucleotides.

In some embodiments, the oligonucleotide may be delivered to cells in combination with a transmembrane delivery system. In some embodiments, this transmembrane delivery system includes lipids, viral vectors, etc. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that does not require any delivery agent. In some embodiments, the method includes using a transmembrane delivery system to deliver siRNA or sdRNA oligonucleotides to a TIL population.

The oligonucleotides and oligonucleotide compositions are contacted (e.g., brought into contact, also referred to herein as being administered or delivered to) and taken up, including passively via the TIL. sdRNA may be added to the TIL as described herein at the following times: during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D), after step D and before collection in step E, during or after collection in step F, before or during final formulation and/or transfer to the infusion bag in step F, and before any optional cryopreservation step in step F. Furthermore, sdRNA may be added after thawing from any cryopreservation step in step F. In some embodiments, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TIL and other agents at concentrations selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM. In some implementations, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TILs and other agents in amounts selected from the group consisting of: 0.1 μM sdRNA/10,000 TILs/100 μL of medium, 0.5 μM sdRNA/10,000 TILs/100 μL of medium, 0.75 μM sdRNA/10,000 TILs/100 μL of medium. IL/100μL medium, 1μM sdRNA/10,000 TILs/100μL medium, 1.25μM sdRNA/10,000 TILs/100μL medium, 1.5μM sdRNA/10,000 TILs/100μL medium, 2μM sdRNA/10,000 TILs/100μL medium, 5μM sdRNA/10,000 TILs/100μL medium, or 10μM sdRNA/10,000 TILs/100μL medium. In some implementations, one or more sdRNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to TIL cultures twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the pre-REP or REP phase.

The oligonucleotide compositions of the present invention, including siRNA or sdRNA, can be contacted with TILs as described herein during an amplification process, for example, by dissolving high concentrations of siRNA or sdRNA in a cell culture medium and allowing sufficient time for passive uptake. In some embodiments, the method of the present invention includes contacting a TIL population with the oligonucleotide composition as described herein. In some embodiments, the method includes dissolving an oligonucleotide (e.g., siRNA or sdRNA) in a cell culture medium and contacting the cell culture medium with the TIL population. TILs may be a first, second, and/or third population as described herein.

In some embodiments, delivery of oligonucleotides into cells can be enhanced by suitable methods known in the art, including calcium phosphate, DMSO, glycerol or polydextrose, electroporation, or by transfection, for example using cationic, anionic or neutral lipid compositions or liposomes, using methods known in the art, such as those described in: U.S. Patent Nos. 4,897,355; 5,459,127; 5,631,237; 5,955,365; 5,976,567; 10,087,464; and 10,155,945; and Bergan et al., Nucl. Acids Res. 1993, 21,3567, the disclosures of which are each incorporated herein by reference.

In some embodiments, more than one siRNA or sdRNA is used to reduce the expression of a target gene. In some embodiments, one or more of PD-1, TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH that target siRNA or sdRNA are used together. In some embodiments, PD-1 siRNA or sdRNA is used with one or more of TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH to reduce the expression of more than one gene target. In some embodiments, LAG3 siRNA or sdRNA is used in combination with CISH that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, TIGIT siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CISH siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CTLA-4 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, LAG-3 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, CBL-B siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, the siRNA or sdRNA targeting one or more of PD-1, TIM-3, CBL-B, LAG3, and/or CISH described herein is available from Advirna LLC, Worcester, Massachusetts, USA, or from several other suppliers.

In some embodiments, siRNA or sdRNA targets genes selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets genes selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, the siRNA or sdRNA targets genes selected from PD-1, LAG-3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from PD-1 and one of the following: LAG3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets CISH, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets PD-1. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CBL-B.

As discussed herein, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been genetically modified by gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing of TIL populations via nucleotide insertion (RNA or DNA) to promote and inhibit the expression of one or more proteins, and combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into therapeutic populations, wherein the methods include gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, said gene editing techniques being suitable for use according to the present invention. Such methods include those described below as well as viral and transposon methods described elsewhere herein. In some embodiments, methods for genetically modifying TILs, MILs, or PBLs to express CCRs may further include modifications to inhibit gene expression via stable gene knockout or temporary gene knockdown of such genes.

In some embodiments, the method includes methods for genetically modifying TIL populations in a first, second, and/or third population as described herein. In some embodiments, the method for genetically modifying TIL populations includes a step of stabilizing a gene incorporated for producing or repressing (e.g., silencing) one or more proteins. In some embodiments, the method for genetically modifying TIL populations includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, including the step of applying a series of at least three DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time, and such that the survival rate of the TIL is maintained. In some embodiments, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for genetically modifying TIL populations include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population includes a transfection step using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference. The TIL may be a first TIL population, a second TIL population, and/or a third TIL population as described herein.

According to one embodiment, gene editing methods may include the use of programmable nucleases that mediate the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the product of the target gene.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems (e.g., Cas9) target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, the method for amplifying TILs into a therapeutic population can be performed according to any embodiment of the method described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein the method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by in vitro comparison of gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs. In some implementations, the method includes gene editing of TIL populations using CRISPR, TALE, and/or ZFN methods.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs using a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf1). According to a particular embodiment, the CRISPR method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the CRISPR method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

CRISPR stands for clustered regularly spaced short palindromic repeats. The method of gene editing using the CRISPR system is also referred to herein as the CRISPR method. There are three types of incorporating RNA and Cas proteins and CRISPR systems that can be used according to the present invention: type I, II, and III. Type II CRISPR (using Cas9 as an example) is one of the most fully characterized systems.

CRISPR technology is adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins (including Cas9) to prevent attacks from viruses and other foreign organisms by cleaving and destroying the DNA of invading foreign organisms. CRISPR is a DNA-specialized region with two unique characteristics: nucleotide repeat sequences and spacers. The nucleotide repeat sequences are distributed throughout the CRISPR region, with short foreign DNA segments (spacers) interspersed within the repeat sequences. In the type II CRISPR/Cas system, the spacers are integrated into the CRISPR genomic locus and transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNA (tracrRNA) and guide the Cas proteins to perform sequence-specific cleavage and silence pathogenic DNA. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved protospacer adjacent motif (PAM) sequence containing a dinucleotide upstream of the crRNA-binding region. Thus, the CRISPR/Cas system can retarget to cleave almost any DNA sequence by redesigning the crRNA. The crRNA and tracrRNA in the native system can be simplified to a single guide RNA (sgRNA) of approximately 100 nucleotides for genetic engineering. The CRISPR/Cas system can be directly introduced into human cells by co-delivering plasmids expressing the Cas9 endonuclease and the essential crRNA component. Different Cas protein variants can be used to reduce targeting limitations (e.g., heterologous homologs of Cas9, such as Cpf1).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via CRISPR methods include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. , CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using CRISPR methods and applicable to embodiments of the invention are described in U.S. Patents 8,697,359, 8,993,233, 8,795,965, 8,771,945, 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 8,871,445, 8,906,616, and 8,895,308, the disclosures of which are each incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are available from companies such as GenScript.

In some implementations, genetic modification of the TIL population as described herein can be performed using a CRISPR/Cpf1 system as described in U.S. Patent No. US9790490, the disclosure of which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the TALE method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the TALE method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for transcription activator-like effector protein, which includes transcription activator-like effector nucleases (TALENs). The method of gene editing using the TALE system is also referred to herein as the TALE approach. TALEs are naturally occurring proteins from the plant pathogenic bacterium *Xanthomonas* and contain a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize consecutive DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by a 14–20 base-pair spacer region, pull FokI monomers together to dimerize and generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are each incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a zinc finger or zinc finger nuclease method. According to a particular embodiment, the zinc finger method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the zinc finger method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Individual zinc fingers, exhibiting a conserved ββα conformation, contain approximately 30 amino acids. Several amino acids on the α-helix surface typically contact the 3 bp in the main groove of the DNA at varying levels of selectivity. Zinc fingers possess two protein domains. The first domain is a DNA-binding domain, which includes eukaryotic transcription factors and contains the zinc finger. The second domain is a nuclease domain, which includes the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

Individual zinc finger (ZFN) DNA-binding domains typically contain between three and six individual zinc finger repeats and can each recognize between nine and 18 base pairs. If the zinc finger domains are specific for their intended target sites, even a pair of three-finger ZFNs recognizing a total of 18 base pairs can theoretically target a single locus in the mammalian genome. One method for generating novel zinc finger arrays is to combine smaller zinc finger “modules” with known specificity. The most common module assembly process involves combining three separate zinc fingers, each recognizing a 3-base-pair DNA sequence, to produce a three-finger array recognizing a 9-base-pair target site. Alternatively, selection-based methods, such as oligopool engineering (OPEN), can be used to select new zinc finger arrays from a randomly grouped library that considers context-dependent interactions between neighboring fingers. Engineered zinc fingers are available from Sangamo Biosciences (Richmond, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the zinc finger method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, and C. ASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6 R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the zinc finger method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which can be used according to embodiments of the invention are described in U.S. Patents 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, each of which is incorporated herein by reference.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which may be used according to other embodiments of the invention are described in Beane et al., Mol. Therapy, 2015, 23, 1380-1390, the disclosure of which is incorporated herein by reference.

In some embodiments, TILs are optionally genetically engineered to include other functionalities, including (but not limited to) high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). In some embodiments, the method includes genetically engineering a population of TILs to include high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). Suitablely, the TIL population may be a first, second, and/or third population as described herein.

D. Closed systems used in TIL manufacturing

This invention provides a closed system for use during TIL (Total Intake Liquid) culture. Such a closed system allows for the prevention and/or reduction of microbial contamination, allows for the use of fewer culture flasks, and allows for lower costs. In some embodiments, the closed system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

A sterile connection device (STCD) creates a sterile weld between two compatible tubes. This procedure allows for the aseptic connection of multiple containers and tube diameters. In some embodiments, the closure system includes a Luer lock and a heat-sealing system as described in the examples. In some embodiments, the closure system is introduced under aseptic conditions via a syringe to maintain the sterility and closure properties of the system. In some embodiments, a closure system as described in the examples is used. In some embodiments, the TIL is formulated into a final product formulation container according to the method described in the examples herein.

In some embodiments, the closed system uses a single container from the time the tumor fragment is obtained until it is ready for TIL administration to the patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G container, and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. The closed system or closed TIL cell culture system is characterized by tightly sealing the system from the outside to create a closed environment free from bacterial, fungal, and/or other microbial contamination once the tumor sample and/or tumor fragment has been added.

In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

The closed system allows TILs to grow in the absence of microbial contamination and/or with significantly reduced microbial contamination.

Furthermore, the pH, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment each change with cell culture. Therefore, even if the culture medium suitable for cell culture is circulated, the closed environment still needs to be continuously maintained to provide the optimal environment for TIL proliferation. For this purpose, it is desirable to monitor the physical factors of pH, carbon dioxide partial pressure, and oxygen partial pressure within the culture medium of the closed environment using sensors. These signals are used to control a gas exchanger installed at the inlet of the culture environment, and the gas partial pressure of the closed environment is adjusted in real time according to changes in the culture medium to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system incorporating a gas exchanger equipped with monitoring devices for measuring the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment at the inlet, and optimizing the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring devices.

In some implementations, the pressure within the closed environment is controlled continuously or intermittently. That is, the pressure within the closed environment can be varied by means of, for example, a pressure maintaining device, thereby ensuring that the space is suitable for TIL growth under positive pressure or promoting exudation of fluid and thus promoting cell proliferation under negative pressure. Furthermore, by intermittently applying negative pressure, it is possible to uniformly and effectively displace the circulating fluid within the closed environment by temporarily reducing its volume.

In some implementations, the optimal culture components for TIL proliferation may be replaced or added, and factors including, for example, IL-2 and/or OKT3, and combinations thereof may be added.

E.TIL's optional cryopreservation

Optionally, the primary TIL population (e.g., a second TIL population) or an amplified TIL population (e.g., a third TIL population) can be cryopreserved. In some embodiments, therapeutic TIL populations are cryopreserved. In some embodiments, cryopreservation occurs on TILs collected after a second amplification. In some embodiments, TILs are cryopreserved in exemplary step F of Figures 1 and/or 8 (particularly, e.g., Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs are cryopreserved in infusion bags. In some embodiments, TILs are cryopreserved before being placed in infusion bags. In some embodiments, TILs are cryopreserved without being placed in infusion bags. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is typically accomplished by placing a population of TILs in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are then placed in cryopreservation vials and stored at -80°C for 24 hours, with optional transfer to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some embodiments, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLifeSolutions). In some embodiments, the TIL population is cryopreserved using cryopreservation medium containing dimethyl sulfoxide (DMSO). In some embodiments, the TIL population is cryopreserved using a 1:1 (vol:vol) ratio of CS10 to cell culture medium. In some embodiments, the TIL population is cryopreserved using approximately a 1:1 (vol:vol) ratio of CS10 to cell culture medium (also containing additional IL-2).

As discussed above and illustrated in steps A through E in Figures 1 and/or 8 (especially, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P), cryopreservation can occur at multiple points in the TIL amplification process. In some embodiments, the expanded TIL population after the first amplification (as provided, for example, according to step B) or after one or more second amplifications according to step D of Figure 1 or Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be cryopreserved. Cryopreservation is typically accomplished by placing the TIL population in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are placed in cryopreservation vials and stored at -80°C for 24 hours, wherein optionally they are transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, TILs are cryopreserved according to the method provided in Example 6.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some cases, the TIL populations of step B in Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be immediately cryopreserved using the schemes discussed below. Alternatively, the main TIL population may undergo steps C and D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), and then cryopreservation following step D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Similarly, in cases where genetically modified TILs will be used in a therapy, the TIL population from step B or step D of Figure 1 or Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) may undergo genetic modification for appropriate treatment.

F. Phenotypic characteristics of amplified TILs

In some embodiments, the expression of TILs after amplification is analyzed using multiple phenotypic markers, including those described herein and in the examples. In some embodiments, the expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characterization of TILs is analyzed after the first amplification in step B of FIG1 or FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transition in step C from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transformation from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and after cryopreservation. In some implementations, the phenotypic characteristics of TIL are analyzed after the second amplification in step D from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed after two or more amplifications from step D of FIG1 or FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P).

In some implementations, the marker is selected from the group consisting of CD8 and CD28. In some implementations, the expression of CD8 is examined. In some implementations, the expression of CD28 is examined. In some implementations, compared to other processes (e.g., the Gen 3 process provided in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B, the expression of CD28 is examined. The 2A process provided in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) produces a higher expression of CD8 and/or CD28 on the TIL according to the process of the present invention. In some implementations, the expression of CD8 on the TIL produced by the process according to the invention is higher than that of other processes (e.g., the Gen 3 process provided in, for example, FIG8 (especially, for example, FIG8B)) and the 2A process provided in, for example, FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, the expression of CD28 on the TIL produced by the process according to the invention is higher than that of the 2A process provided, such as, for example, in Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to other processes (e.g., Figure 8A and/or Figure 2A, for example, Figure 8A). In some embodiments, high CD28 expression indicates a younger and more durable TIL phenotype. In some embodiments, the expression of one or more regulatory markers is measured.

In some implementations, during any step of the method for amplifying tumor-infiltrating lymphocytes (TILs) as described herein, no selection of a first TIL population, a second TIL population, a third TIL population, or a collected TIL population is made based on CD8 and/or CD28 expression.

In some embodiments, the percentage of central memory cells in the TIL produced by the method according to the invention is higher than that of process 2A, as provided in, for example, Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the memory markers of the central memory cells are selected from the group consisting of CCR7 and CD62L.

In some implementations, the CD4+ and/or CD8+ TIL memory subsets may be divided into different memory subsets. In some implementations, the CD4+ and/or CD8+ TILs contain initial (CD45RA+CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain central memory (CM; CD45RA-CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain effect memory (EM; CD45RA-CD62L-) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain RA+ effect memory/effect (TEMRA/TEFF; CD45RA+CD62L+) TILs.

In some embodiments, the TIL expresses one or more markers selected from the group consisting of granzyme B, perforin, and granulosin. In some embodiments, the TIL expresses granzyme B. In some embodiments, the TIL expresses perforin. In some embodiments, the TIL expresses granulosin.

In some embodiments, a cytokine release assay may also be used to evaluate cytokine release from restimulated TILs. In some embodiments, interferon-γ (IFN-γ) secretion from TILs may be evaluated. In some embodiments, IFN-γ secretion is measured by an ELISA assay. In some embodiments, IFN-γ secretion is measured by an ELISA assay after a rapid second amplification step, following step D as provided, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL health is measured by IFN-γ (IFN-γ) secretion. In some embodiments, IFN-γ secretion indicates active TILs. In some embodiments, a potency assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the culture medium of TILs stimulated with antibodies against CD3, CD28, and CD137/4-1BB. The IFN-γ level in the culture medium from these stimulated TILs can be determined by measuring IFN-γ release. In some embodiments, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), step D in the Gen 3 process provided in the TIL shows increased IFN-γ production compared to step D in the 2A process provided, for example, Figure 8 (especially, for example, Figure 8A), indicating an increased cytotoxic potential of the TIL in step D. In some embodiments, IFN-γ secretion is increased by one, two, three, four, or five times or more. In some embodiments, IFN-γ secretion is increased by one time. In some embodiments, IFN-γ secretion is increased by two times. In some embodiments, IFN-γ secretion is increased by three times. In some embodiments, IFN-γ secretion is increased by four times. In some embodiments, IFN-γ secretion is increased by five times. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ in ex vivo TILs is measured. In some embodiments, IFN-γ in ex vivo TILs is measured, including TILs produced by the methods of the present invention (including, for example, the method of Figure 8B).

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL to about 1000 pg/mL or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some implementations, it is capable of secreting at least 200 pg/mL, at least 250 pg/mL, at least 300 pg/mL, at least 350 pg/mL, at least 400 pg/mL, at least 450 pg/mL, at least 500 pg/mL, at least 550 pg/mL, at least 600 pg/mL, at least 650 pg/mL, at least 700 pg/mL, at least 750 pg/mL, at least 800 pg/mL, at least 850 pg/mL. L, at least 900 pg/mL, at least 950 pg/mL, or at least 1000 pg/mL or more of IFN-γ TILs are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL/5e5 cells to about 1000 pg/mL/5e5 cells or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, it is capable of secreting at least 200 pg/mL/5e5 cells, at least 250 pg/mL/5e5 cells, at least 300 pg/mL/5e5 cells, at least 350 pg/mL/5e5 cells, at least 400 pg/mL/5e5 cells, at least 450 pg/mL/5e5 cells, at least 500 pg/mL/5e5 cells, at least 550 pg/mL/5e5 cells, at least 600 pg/mL/5e5 cells, at least 650 pg/mL/5e5 cells, at least 700 pg/mL/5e5 cells, and at least 750 pg/mL/5e5 cells. TILs of IFN-γ produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) are TILs produced by at least 800 pg/mL/5e5 cells, at least 850 pg/mL/5e5 cells, at least 900 pg/mL/5e5 cells, at least 950 pg/mL/5e5 cells, or at least 1000 pg/mL/5e5 cells or more. In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that express and increase the diversity of T cell reservoirs. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method called Gen2 exemplified in Figure 8 (particularly, for example, Figure 8A). In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased. In some embodiments, processes as described herein (e.g., the Gen 3 process) exhibit higher clonal diversity based on the number of unique peptide CDRs within the sample compared to other processes (e.g., the Gen 2 process).

In some embodiments, TIL activation and depletion can be determined by examining one or more biomarkers. In some embodiments, activation and depletion can be determined using multicolor flow cytometry. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: CD3, PD-1, 2B4/CD244, CD8, CD25, BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183, CD69, CD95, CD127, CD103, and/or LAG-3. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, PD-1, TIGIT, and/or TIM-3. In some embodiments, activation and depletion of markers include (but are not limited to) one or more markers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, CD103+/CD69+, CD103+/CD69-, PD-1, TIGIT, and/or TIM-3. In some embodiments, T cell markers (including activation and depletion markers) may be identified and/or analyzed to examine T cell activation, suppression, or function. In some embodiments, T cell markers may include (but are not limited to) one or more markers selected from the group consisting of: TIGIT, CD3, FoxP3, Tim-3, PD-1, CD103, CTLA-4, LAG-3, BTLA-4, ICOS, Ki67, CD8, CD25, CD45, CD4, and/or CD59.

In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, TILs exhibiting secretion of granzyme B at levels higher than 1000 pg/mL to 300000 pg/mL or more are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed at levels above 1000 pg/mL, above 2000 pg/mL, above 3000 pg/mL, above 4000 pg/mL, above 5000 pg/mL, above 6000 pg/mL, above 7000 pg/mL, above 8000 pg/mL, above 9000 pg/mL, above 10000 pg/mL, above 20000 pg/mL, above 30000 pg/mL, above 40000 pg/mL, above 50000 pg/mL, and above 60000 pg/mL. TILs with granzyme B concentrations of 0000 pg/mL, above 70000 pg/mL, above 80000 pg/mL, above 90000 pg/mL, above 100000 pg/mL, or more are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 1000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 2000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 3000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 4000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 5000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 6000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 7000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 8000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 9000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 10,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 20,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 30,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 40,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 50,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 60,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 70,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 80,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 90,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 100,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of granzyme B above 260,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, the amplification method of the present invention produces an amplified TIL population exhibiting increased in vitro granzyme B secretion compared to a non-amplified TIL population, including, for example, the TILs provided in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased by at least one to fifty times or more compared to a non-amplified TIL population. In some embodiments, IFN-γ secretion is increased by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least thirty, at least forty, at least fifty times or more compared to a non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least doubled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least quadrupled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least five times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least six times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least seven times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least eight times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least nine-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least ten-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least twenty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least thirty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least forty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least fifty-fold compared to the non-amplified TIL population.

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times lower levels of TNF-α (i.e., TNF-alpha) compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α (i.e., TNF-alpha) are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, IFN-γ and granzyme B levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, granzyme B and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ, granzyme B, and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some implementations, phenotypic characteristics are examined after cryopreservation.

G. Other process implementation plans

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days or about 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days or about 1 to 10 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling-up of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein in the second container, the second TIL population from the small-scale culture is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days or approximately 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring and distributing the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 to 7 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for approximately 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for approximately 1 to 8 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a vertical scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 5 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a longitudinal scaling up of the culture by: (1) performing rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-g500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by contacting a first TIL population with a culture medium that also contains exogenous antigen-presenting cells (APCs), wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the culture medium is supplemented with additional exogenous APC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 2:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, or 2.3:1. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the initial first amplification is exactly or approximately 1× ^10⁸ , ^1.1 ×10⁸, 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, 1.5× ^10⁸ , 1.6 ^{×10⁸ ,} 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9×10⁸, 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , 3.1× ^10⁸ , or 3.1×10⁸. ⁸ , 3.2× ^10⁸ , 3.3× ^10⁸ , 3.4× ^10⁸ , or 3.5× ^10⁸ APCs, wherein the number of APCs added in the rapid second amplification is exactly or approximately 3.5× ^10⁸ , 3.6×10⁸, ^3.7 × ^10⁸ , 3.8× ^10⁸ , 3.9× ^10⁸ , 4×10⁸, 4.1× ^10⁸ , ^4.2 × ^10⁸ , 4.3× ^10⁸ , 4.4× ^10⁸ , 4.5×10⁸, 4.6× ^10⁸ , 4.7× ^10⁸ , 4.8× ^10⁸ , 4.9× ^10⁸ , 5× ^10⁸ , 5.1× ^10⁸ , 5.2× ^{10⁸ ,} or 5.3× ^10⁸. 5.4× ^10⁸ , 5.5× ^10⁸ , 5.6×10⁸, 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2×10⁸, 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , ^6.6 × ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , ^7.1 × ^10⁸ , 7.2×10⁸, 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , ^7.8 × ^10⁸ 7.9× ^10⁸ , 8× ^10⁸ , 8.1×10⁸, 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7×10⁸, 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , ^9.6 × ^10⁸ , 9.7× ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or} 1× ^10⁹ APCs.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1 × ^10⁸ APCs to exactly or about 3.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 3.5 × ^10⁸ APCs to exactly or about 1 × ^10⁹ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 2 × ^10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or approximately 2.5 × ^10⁸ APCs are added to an initial first amplification, and exactly or approximately 5 × ^10⁸ APCs are added to a rapid second amplification.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein multiple tumor fragments are distributed into multiple separate containers, in each separate container a first TIL population is obtained in step (a), a second TIL population is obtained in step (b), and a third TIL population is obtained in step (c), and therapeutic TIL populations from the multiple containers in step (c) are combined to produce a collected TIL population from step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of tumors are uniformly distributed into a plurality of separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise at least two separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to twenty separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to fifteen separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to ten separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to five separate containers.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the plurality of separate containers comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first amplification of a first TIL population in step (b) is performed in each container, and a rapid second amplification of a second TIL population generated from such a first TIL population is performed in the same container as in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each of the separate containers includes a first breathable surface area.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein multiple tumor fragments are distributed in a single container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a single container includes a first breathable surface region.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in a second container containing a second breathable surface region.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second container is larger than the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the second breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:9.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:2.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected as exactly or greater. Approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4... 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 1.5:1 to exactly or about 100:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 50:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 25:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 50 times larger in number than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times larger than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is supplemented with additional IL-2 exactly or about 2 days or exactly or about 3 days after the start of the second time period in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, further comprising the step of cryopreserving the collected TIL population in step (d) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, including an additional step (e) after step (d) of transferring the collected TIL population from step (d) to an infusion bag optionally containing HypoThermosol.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, comprising the step of cryopreserving an infusion bag containing the collected TIL population in step (e) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation process is performed using a 1:1 ratio of collected TIL populations to cryopreservation medium.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (b) is 2.5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (c) is 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a PBMC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are artificial antigen-presenting cells.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a membrane-based cell processing system is used for the collection in step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the collection in step (d) is performed using the LOVO cell processing system.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 5 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 10 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 15 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 20 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 25 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 30 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 35 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 40 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 45 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or about 50 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 segments per container.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately 27 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 20 ^mm³ to exactly or about 50 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 21 ^mm³ to exactly or about 30 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 22 ^mm³ to exactly or about 29.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 23 ^mm³ to exactly or about 29 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 24 ^mm³ to exactly or about 28.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 25 ^mm³ to exactly or about 28 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 26.5 ^mm³ to exactly or about 27.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately the following: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 30 to exactly or about 60 segments, wherein the total volume is exactly or about 1300 ^mm³ to exactly or about 1500 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total volume is exactly or about 1350 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total mass is exactly or about 1 gram to exactly or about 1.5 grams.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is provided in a container in the form of a G container or a Xuri cell bag.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is from about 10,000 IU/mL to about 5,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is about 6,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second time period in step (c) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 5 days, 6 days, or 7 days, respectively.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 16 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 17 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 16 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 14 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 15 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 14 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 15 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 14 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 15 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 16 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 18 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the therapeutic TIL population collected in step (d) contains a therapeutically effective dose of TIL sufficient for use with TIL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of TILs sufficient for a therapeutically effective dose is exactly or about 2.3 × 10¹⁰ to exactly or about 13.7 × 10¹⁰.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein effector T cells and/or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression relative to effector T cells and/or central memory T cells obtained from the second cell population in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a closed container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a G container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-100.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-500.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared by the methods described in any of the foregoing paragraphs as applicable above.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs) or OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of antigen-presenting cells (APCs) and OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 16 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 17 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 18 days.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased interferon-γ production.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased polyclonalness.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased efficacy.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A through F above, or according to steps A through F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than one times the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than twice the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least three times more interferon-γ.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any antigen-presenting cells (APCs). In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are made capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that is capable of producing at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are able to produce at least one times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least three times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy), a core needle biopsy, a core needle puncture biopsy, or a fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a fine-needle aspirate.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is obtained by core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirates; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first amplification step; (iii) the method includes a period of approximately 8 days for initiating the first amplification; and (iv) the method includes a period of approximately 11 days for rapid second amplification. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating the first expansion step; (iii) the method includes a period of approximately 8 days for initiating the first expansion; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing the subcultures for approximately 6 days. In some of the foregoing embodiments, up to 5 subcultures are cultured separately in containers of the same size or larger as the container in which the second TIL population is initially cultured in the rapid second expansion. In some of the foregoing embodiments, the culture of the second TIL population is evenly distributed among up to 5 subcultures. In some of the aforementioned embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1 to about 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step to obtain a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days; and (iv) the method includes a rapid second expansion step by culturing the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 11 days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days to obtain a second TIL population; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing each of the subcultures in a cell culture medium containing IL-2 for approximately 6 days. In some of the foregoing embodiments, up to five subcultures are cultured in containers of the same size or larger than the container in which the second TIL population was started during the rapid second amplification. In some of the foregoing embodiments, the cultures of the second TIL population are evenly distributed among up to five subcultures. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method comprises obtaining a first TIL population from 1 to about 10 tumor tissue samples from a subject by core needle biopsy; (ii) the method comprises, prior to performing the first amplification step, culturing the first TIL population in a G-REX-100M culture flask in a cell culture medium containing 0.5 L CM1 medium of 6000 IU IL-2/mL for about 3 days; (iii) the method comprises initiating the first amplification by adding 0.5 L CM1 medium containing 6000 IU/mL IL-2, 30 ng/mL OKT-3, and about ^10⁸ feeder cells, and culturing for about 8 days; and (iv) the method comprises performing a rapid second amplification by transferring the second TIL population to a 5 L CM1 medium containing 3000 IU/mL IL-2, 30 ng/mL OKT-3, and 5 × ^10⁹ feeder cells. (a) The culture is divided into up to five subcultures by transferring ^10⁹ TILs into each of up to five G-REX-500MCS culture flasks containing 5 L AIM-V medium with 3000 IU/mL IL-2, and said subcultures are cultured for approximately six days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (a) initiating a first expansion of a first T cell population obtained from a donor by culturing a first T cell population to achieve growth and initiate activation of the first T cell population; (b) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (c) collecting the second T cell population. In other embodiments, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling up of culture scale by: (a) rapidly expanding the first T cell population by culturing it in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second expansion step is broken down into multiple steps to achieve a longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of up to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), the first T cell population is cultured in a first culture medium containing OKT-3 and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises a 4-1BB agonist, OKT-3, and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the first T cell population is cultured in a second culture medium containing OKT-3, IL-2 and antigen-presenting cells (APC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains 4-1BB agonist, OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs in the second APC group to the number of APCs in the first APC group is about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs in the first APC group is about 2.5 × ^10⁸ and the number of APCs in the second APC group is about 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is laminated onto a first breathable surface with an average thickness of two APC layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC group is laminated onto the first breathable surface with an average thickness selected from 4 to 8 APC layers.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the average number of APC layers stacked on the first breathable surface in step (b) to the average number of APC layers stacked on the first breathable surface in step (a) is 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 2.0 × ^10⁶ APCs/ ^cm² to just or about 3.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 2.5 × ^10⁶ APCs/ ^cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 2.0 × ^10⁶ APCs/ ^cm² to exactly or about 3.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a peripheral blood mononuclear cell (PBMC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMCs are irradiated and the donor for the first T cell population is exogenous.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are bone marrow infiltrating lymphocytes (MIL).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are peripheral blood lymphocytes (PBLs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by separation from whole blood of a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by isolating from the donor's apheresis product.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first T cell population is isolated from whole blood or apheresis products of a donor by positive or negative selection of T cell phenotype.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cell phenotype is CD3+ and CD45+.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein T cells are isolated from NK cells prior to initiating a first expansion of a first T cell population. In other embodiments, T cells in a first T cell population are separated from NK cells by removing CD3-CD56+ cells from the first T cell population. In other embodiments, CD3-CD56+ cells are removed from the first T cell population by using a gating strategy that removes CD3-CD56+ cell fractions and recovers negative fractions. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of NK cells. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of CD3-CD56+ cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of NK cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of NK cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with ovarian cancer.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or about 1 × ^10⁷ T cells from a first T cell population are seeded into a container to initiate an initial first expansion culture in such a container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein a first T cell population is distributed into a plurality of containers, and exactly or about 1 × 10⁷ T cells from the first T cell population are seeded in each container to initiate an initial first expansion culture in such containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second T cell population collected in step (c) is a therapeutic TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via small biopsy (including, for example, perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a donor, including small biopsies (such as perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsy of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a donor.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsies, core needle biopsies, or needle biopsies obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the initiation of the first expansion is performed in a container containing a first breathable surface region, wherein the initiation of the first expansion is performed for a first time period of approximately 7 or 8 days to obtain the second TIL population. (iii) A second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iv) a rapid second expansion is performed by supplementing the second TIL population with a second cell culture medium containing additional IL-2, OKT-3 and APC to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (ii), wherein the rapid second expansion is performed for a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container containing a second ventilated surface area; (v) the therapeutic TIL population obtained from step (iii) is collected; and (v) the collected TIL population from step (iv) is transferred to an infusion bag.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsy, core needle biopsy, or needle biopsy samples obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; and (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a second TIL population, wherein the initiation... The first amplification is carried out over a first period of approximately 7 or 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iii) a rapid second amplification is carried out by contacting the second TIL population with a third cell culture medium containing IL-2, OKT-3 and APC to produce a third TIL population, wherein the rapid second amplification is carried out over a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second amplification is carried out in a container containing a second ventilated surface area; and (iv) the therapeutic TIL population obtained from step (iii) is collected.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with an additional amount of third culture medium and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with a fourth medium containing IL-2 and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the culture is split into up to five subcultures after day 5 of the second time period.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein all steps in the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (i) initiating a first expansion of a first T cell population by culturing a first T cell population to achieve growth and initiate activation of the first T cell population, the first T cell population being derived from a tumor sample obtained from one or more small biopsies, core needle biopsies, or needle biopsies of a tumor in a donor; (ii) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population a second T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (iv) collecting the second T cell population. In some embodiments, the tumor sample is obtained from multiple core needle biopsies. In some embodiments, the multiple core needle biopsies are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, and 10 core needle biopsies.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein T cells or TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubating the tumor in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, California). In some embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes one or more dissociation (digestive) enzymes, such as (but not limited to) collagenase (including any blended or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersant enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, protease type XIV (streptomycin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof. In other embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes collagenase (including any blended or type of collagenase), neutral protease (dispersant enzyme), and deoxyribonuclease I (DNase).

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a TIL population undergoes a gene-editing process. In some embodiments, the gene-editing process may be performed at any time during the TIL amplification process, including before, during, or after any step of the amplification process, such as before, during, or after one or more of steps A, B, C, D, E, and F outlined above.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein an activation step follows a first amplification step. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and/or anti-CD38 beads for about 1-7 days. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and anti-CD38 beads for about 1-7 days. In some embodiments, the activation step is performed for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the activation step is followed by a gene editing step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a gene editing step follows a first amplification step. In some embodiments, when the first amplification step includes OKT-3, a gene editing step follows the first amplification step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the gene editing step includes a sterile electroporation step on a TIL population. In some embodiments, the sterile electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates the expression of PD-1. According to some embodiments, the gene editor further comprises a TALE nuclease system that downregulates the expression of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CTLA-4 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/LAG-3 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CISH double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CTLA-4. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and TIGIT. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and TIGIT. According to some implementation schemes, the obtained TILs showed downregulation of CTLA-4 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CISH expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of TIGIT and CBL-B expression.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a quiescent step follows the gene editing step. In some embodiments, the quiescent step comprises incubating a TIL population at about 30-40°C and about 5% _CO2 for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 30°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1 hour, followed by about 15-23 hours at about 30°C.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a second amplification step follows the resting step.

VIII. Pharmaceutical Compositions, Dosage, and Dosing Regimens

In some embodiments, TILs, MILs, or PBLs (including TILs, MILs, or PBLs genetically modified to express CCR) amplified and/or genetically modified using the methods of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC or melanoma. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is metastatic NSCLC or metastatic melanoma. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of NSCLC. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

In other embodiments, the present invention provides an infusion bag that includes the therapeutic TIL population described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a pharmaceutically acceptable carrier as described in any of the preceding paragraphs.

In other embodiments, the present invention provides an infusion bag comprising the TIL composition described in any of the preceding paragraphs.

In other embodiments, the present invention provides a cryopreservation formulation for a therapeutic TIL population as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a cryopreservation culture medium as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains DMSO.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a cryopreservation formulation of the TIL composition described in any of the preceding paragraphs.

In some embodiments, TILs amplified using the methods of this disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly when the cancer is NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

IX. Methods of treating patients

The treatment method begins with the collection and culture of raw TILs. Such methods have been described in the art, for example, by reference in their entirety to Jin et al., J. Immunotherapy, 2012, 35(3):283-292. The implementation of the treatment method is described throughout the following sections, including examples.

The methods described herein, including, for example, those described in steps A through F above or those described in steps A through F above (and also, for example, those shown in Figures 1, 36, and/or 8), have been found to have specific uses in the treatment of cancer patients (e.g., as described in Goff et al., J. Clinical Oncology, 2016, 34(20):2389-239 and the Supplement, which are incorporated herein by reference in their entirety). In some embodiments, TILs are grown from resected metastatic melanoma remnants as previously described (see Dudley et al., J Immunother., 2003, 26:332-342, which are incorporated herein by reference in their entirety). Fresh tumors can be segmented under sterile conditions. Representative samples can be collected for formal pathological analysis. Single fragments of 2 ^mm³ to ³ mm³ can be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained from each patient. In some embodiments, 24 samples are obtained from each patient. Samples can be placed in individual wells of a 24-well plate, maintained in growth medium containing a high dose of IL-2 (6,000 IU/mL), and tumor destruction and/or TIL proliferation can be monitored. As described herein, any tumor cells remaining viable after treatment can be enzymatically digested into a single-cell suspension and cryopreserved.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumors when available. TILs are considered reactive if overnight co-culture produces interferon-γ (IFN-γ) levels >200 pg/mL and twice the background level. (Goff et al., J Immunother., 2010, 33:840-847; incorporated herein by reference in its entirety). In some embodiments, cultures demonstrating autoreactivity or adequate growth patterns may be selected for secondary amplification (e.g., the secondary amplification provided according to step D of Figures 1, 36, and/or 8), including what is sometimes referred to as rapid amplification (REP). In some embodiments, amplified TILs exhibiting high autoreactivity (e.g., high proliferation during secondary amplification) are selected for additional secondary amplification. In some implementations, TILs with high self-reactivity (e.g., high proliferation during the second amplification provided in step D of Figures 1, 36 and/or 8) are selected for additional second amplification according to step D of Figures 1, 36 and/or 8.

Cell phenotypes of cryopreserved samples of infusion bag TILs can be analyzed by flow cytometry targeting surface markers CD3, CD4, CD8, CCR7, and CD45RA (e.g., FlowJo) (BD BioSciences) and by any of the methods described herein. Serum cytokines were measured using standard enzyme-linked immunosorbent assay (ELISA). An increase in serum IFN-γ was defined as a baseline level >100 pg/mL and greater than 43.

In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, achieve remarkable improvements in clinical efficacy. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, exhibit improved clinical efficacy compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8). In some embodiments, methods other than those described herein include methods referred to as Process 1C and/or Gen 1. In some embodiments, the increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1 or 36, exhibit similar response time and safety profiles compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8).

In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of a subject treated with TIL indicates active TIL. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TIL of a subject treated with TIL prepared by the methods of the present invention, including, for example, those described in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TIL produced by the methods of the present invention. In some embodiments, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or compared to patients treated with TIL prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is quadrupled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is increased fivefold compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the serum of subjects treated with TILs prepared using the methods of the present invention, including those prepared, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy in treating tumors.

In some embodiments, the TILs prepared by the methods of the present invention include those described, for example, in Figure 1 or Figure 36, where IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of subjects treated with TILs indicates active TILs. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TILs of subjects treated with TILs prepared by the methods of the present invention, including methods described, for example, in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TILs produced by the methods of the present invention. In some implementations, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, and/or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, and/or 8, such as those referred to as the process 1C method). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, those other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

Measures of efficacy may include disease control rate (DCR) and overall response rate (ORR), as known in the art and described herein.

A. Methods of treating cancer

The compositions and methods described herein can be used in a method of treating a disease. In some embodiments, they are used to treat hyperproliferative conditions, such as cancer, in adult or pediatric patients. They can also be used to treat other conditions as described herein and in the following paragraphs.

In some implementations, hyperplasia is defined as cancer. In some implementations, hyperplasia is defined as solid tumor cancer. In some implementations, solid tumor cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papillomavirus (HPV), central nervous system-related cancers (including ependymoma, neuroblastoma, neuroblastoma, pineal blastoma, and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSC)). C) Hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, and pharyngeal cancer; kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC), metastatic NSCLC, and small cell lung cancer); melanoma (including uveal melanoma, choroidal melanoma, ciliary body melanoma, iris melanoma, or metastatic melanoma); mesothelioma (including malignant pleural mesothelioma); ovarian cancer; pancreatic cancer (including pancreatic duct adenocarcinoma); penile cancer; rectal cancer; kidney cancer; renal cell carcinoma; sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas); thyroid cancer (including anaplastic thyroid cancer); uterine cancer and vaginal cancer.

In some embodiments, the hyperplastic condition is a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from the group consisting of: chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-B, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified MIL or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-BR, wherein the cancer is a hematologic malignancy.

In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, that is relapsed or refractory to treatment with at least one prior therapy (including chemotherapy, radiotherapy, or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least two prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least three prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy).

In some embodiments, the cancer is a microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. MSI-H and dMMR cancers and their detection have been described in Kawakami et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosure of which is incorporated herein by reference.

In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a non-human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a companion animal.

In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors and/or MEK inhibitors. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method of treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, enrafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof; and difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, bemetinib, selmetinib, pimatetinib, refatinib, and pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is a pediatric cancer.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is uveal melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the uveal melanoma is a choroidal melanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is neuroblastoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a central nervous system (CNS) related cancer. In some embodiments, the pediatric cancer is difficult to treat with chemotherapy. In some embodiments, the pediatric cancer is difficult to treat with radiation therapy. In some embodiments, the pediatric cancer is difficult to treat with dinutuximab.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the CNS-related cancer is neuroblastoma, pinealoblastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein can be used to treat cancer that is refractory to anti-PD-1 or anti-PD-L1 antibodies or resistant to prior treatment. In some embodiments, the patient is a primary refractory patient to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has not shown a prior response to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has shown a prior response to anti-PD-1 or anti-PD-L1 antibodies, followed by cancer progression. In some embodiments, the cancer is refractory to combination therapy with anti-CTLA-4 antibodies and/or anti-PD-1 or anti-PD-L1 antibodies in combination with at least one chemotherapeutic agent. In some embodiments, the prior chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and/or cisplatin. In some prior embodiments, the chemotherapeutic agent is a platinum-based dual chemotherapeutic agent. In some embodiments, the platinum dual therapy comprises a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel, docetaxel, or nab-paclitaxel). In some embodiments, the platinum dual chemotherapeutic agent is combined with pemetrexed.

In some implementations, NSCLC is PD-L1 negative and/or comes from patients with cancers that express PD-L1 and have a tumor proportion score (TPS) <1%, as described elsewhere in this article.

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or anti-PD-L1 antibody, pemetrexed, and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some embodiments, NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient has not received treatment. In some embodiments, NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, NSCLC has been previously treated with a chemotherapy agent. In some embodiments, NSCLC has been previously treated with a chemotherapy agent but is no longer being treated with said chemotherapy agent. In some embodiments, the NSCLC patient has not received anti-PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the NSCLC patient has not received NSCLC treatment or has received chemotherapy agent treatment but has not received PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has not received treatment or has received chemotherapy agent treatment but has not received anti-PD-1/PD-L1 treatment and has low PD-L1 expression. In some embodiments, the NSCLC patient had massive disease at baseline. In some embodiments, the subject has massive disease and low PD-L1 expression at baseline. In some embodiments, the NSCLC patient does not have detectable PD-L1 expression. In some embodiments, the NSCLC patient is untreated or has received chemotherapy but not anti-PD-1/PD-L1 treatment and does not have detectable PD-L1 expression. In some embodiments, the patient has massive disease and does not have detectable PD-L1 expression at baseline. In some embodiments, the NSCLC patient has untreated NSCLC or has received chemotherapy (e.g., received chemotherapy agents) but not anti-PD-1/PD-L1 treatment, and the patient has low PD-L1 expression and/or has massive disease at baseline. In some embodiments, massive disease is indicated when the maximum tumor diameter measured in the transverse or coronal plane is greater than 7 cm. In some embodiments, massive disease is indicated when there are swollen lymph nodes with a short axis diameter of 20 mm or greater. In some embodiments, the chemotherapy agents include standard of care treatments for NSCLC.

In some embodiments, PD-L1 expression is determined by a tumor proportion score. In some embodiments, a subject with refractory NSCLC has a tumor proportion score (TPS) of <1%. In some embodiments, a subject with refractory NSCLC has a TPS of ≥1%. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment.

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, or 8, including, for example, methods referred to as Process 1C). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy in cancer treatment. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8).

In some embodiments, PD-L1 expression is determined by tumor proportion scoring using one or more assays as described herein. In some embodiments, the subject or patient with NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the NSCLC tumors have a TPS of ≥1%. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a TPS of ≥1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and a tumor proportion score has been determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment.

In some implementations, NSCLC is NSCLC exhibiting a tumor proportion score (TPS), or the percentage of live tumor cells obtained from the patient prior to anti-PD-1 or anti-PD-L1 therapy, or the percentage of PD-L1 protein showing partial or complete membrane staining at any intensity of less than 1% (TPS<1%). In some implementations, NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, <0.04%, <0.03%, <0.02%, and <0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, and about 0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS between 0% and 1%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.9%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.8%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.7%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.6%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.5%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.4%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.3%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.2%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.1%. TPS can be measured by methods known in the art, such as those described in Hirsch et al., J. Thorac. Oncol. 2017, 12, 208-222, or methods used to determine TPS prior to treatment with pembrolizumab or other anti-PD-1 or anti-PD-L1 therapies. Methods approved by the U.S. Food and Drug Administration for measuring TPS can also be used. In some embodiments, PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

In some embodiments, partial membrane staining includes 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more. In some embodiments, complete membrane staining includes approximately 100% membrane staining.

In some implementations, testing PD-L1 may involve measuring the level of PD-L1 in a patient's serum. In these implementations, measuring PD-L1 in a patient's serum eliminates the uncertainty of tumor heterogeneity and the discomfort of patients undergoing serial biopsies.

In some implementations, elevated soluble PD-L1 levels compared to baseline or standard levels are associated with a prognostic risk of NSCLC progression. See, for example, Okuma et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli et al., Oncotarget, 2018, 9, 17554-17563. In some implementations, PD-L1 is exosomal PD-L1. In some implementations, PD-L1 is expressed on circulating tumor cells.

In some implementation schemes, the subject or patient has non-small cell lung cancer (NSCLC), characterized by at least one of the following:

i. PD-L1 pre-defined tumor proportion score (TPS) <1%,

ii. The TPS score of PD-L1 is 1%-49%, or

iii. The predetermined absence of one or more driver mutations,

The driving mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20 mutations, KRAS mutations, BRAF mutations, ALK mutations, c-ROS mutations (ROS1 mutations), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and/or altered MET signals, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations, and GNA11 mutations.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population described herein.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition described herein.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to the administration of therapeutically effective doses of the therapeutic TIL population and TIL composition described herein, respectively.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, the method further comprising the step of initiating treatment of the subject with a high-dose IL-2 regimen the day after administration of TIL cells to the subject.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg every eight hours via a 15-minute bolus intravenous infusion until tolerated.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is glioblastoma (including GBM).

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides a therapeutic TIL population as described herein in a method for treating a subject suffering from cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer using a TIL composition described herein, comprising administering a therapeutically effective dose of the TIL composition to the subject.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administration of a therapeutically effective dose of the therapeutic TIL population or TIL composition described herein.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, which further includes the step of starting treatment of the patient with a high-dose IL-2 regimen the day after administration of TIL cells.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg via intravenous infusion every eight hours in the form of 15-minute bolus injections until tolerated.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is glioblastoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides the use of the therapeutic TIL population described herein in a method of treating a subject’s cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides the use of the TIL composition described in any of the foregoing paragraphs in a method of treating a subject’s cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In other embodiments, the present invention provides the use of the therapeutic TIL population or the TIL composition described herein in a method of treating a patient with cancer, the method comprising administering a non-myeloablative lymphocyte depletion regimen to the patient and then administering to the subject a therapeutically effective dose of the therapeutic TIL population or the therapeutically effective dose of the TIL composition described herein in any of the preceding paragraphs.

1. Combination with PD-1 and PD-L1 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, which includes TIL and one or more PD-1 and/or PD-L1 inhibitors.

Programmed death 1 (PD-1) is a 288-amino acid transmembrane immune checkpoint receptor protein expressed by T cells, B cells, natural killer (NK) T cells, activated monocytes, and dendritic cells. PD-1, also known as CD279, belongs to the CD28 family and is encoded by the Pdcd1 gene on chromosome 2 in humans. PD-1 consists of an immunoglobulin (Ig) superfamily domain, a transmembrane domain, and an intracellular domain containing the immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoreceptor tyrosine switching motif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play important roles in immune tolerance, as described by Keir et al., Annu. Rev. Immunol. 2008, 26, 677-704. PD-1 provides a suppressive signal that negatively regulates T cell immune responses. PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273) are expressed on tumor cells and stromal cells, which may encounter activated T cells expressing PD-1, leading to immunosuppression of T cells. PD-L1 is a 290-amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9. Blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2 using PD-1 inhibitors, PD-L1 inhibitors, and/or PD-L2 inhibitors can overcome immune resistance, as demonstrated in recent clinical studies, such as those described in Topalian et al., N. Eng. J. Med. 2012, 366, 2443-54. PD-L1 is expressed on many tumor cell lines, while PD-L2 is primarily expressed on dendritic cells and some tumor lines. In addition to T cells (which induce PD-1 expression upon activation), PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes, and dendritic cells.

In some embodiments, the PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail below. Regarding PD-1 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, references herein to PD-1 inhibitors as antibodies may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, references herein to PD-1 inhibitors may also refer to small molecule compounds or their pharmaceutically acceptable salts, esters, solvates, hydrates, cocrystals, or prodrugs.

In some embodiments, the PD-1 inhibitor is an antibody (i.e., an anti-PD-1 antibody) or a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In some embodiments, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competitively binds to PD-1 and/or binds to an epitope on PD-1. In some embodiments, the antibody competitively binds to PD-1 and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a KD of about 100 pM or less, at a KD of about 90 pM or less, at a KD of about 80 pM or less, at a KD of about 70 pM or less, at a KD of about 60 pM or less, at a KD of about 50 pM or less, at a KD of about 40 pM or less, at a KD of about 30 pM or less, at a KD of about 20 pM or less, at a KD of about 10 pM or less, or at a KD of about 1 pM or less.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a k _{-association rate} of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about ⁸ × 10⁵ l/M·s or faster, at a k _{-association rate} of about 8.5 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9.5 × ^10⁵ l/M·s or faster, or at a k- _{association rate} of about 1 × ^10⁶ l/M·s or faster.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, or with a k _{-dissociation rate} of about 2.1 × ^10⁻⁵ 1/s or slower, or with a k-dissociation rate of about 2.2 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.3 × 10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.6 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.7 × ^10⁻⁵ 1/s or slower. It binds to human PD-1 at a k- _{dissociation rate} of 1/s or slower, at a k- _{dissociation rate} of approximately 2.8 × ^10⁻⁵ 1/s or slower, at a k- _{dissociation rate} of approximately 2.9 × ^10⁻⁵ 1/s or slower, or at a k- _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, or about 6 nM or less. The binding of human PD-L2 to human PD-1; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower; or blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-1 inhibitor is nivolumab (available from Bristol-Myers Squibb via OPDIVO) or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-1 receptor. In some embodiments, the anti-PD-1 antibody is an immunoglobulin G4κ anti-(human CD274) antibody. Nivolumab is designated Chemical Abstracts Service (CAS) Registry No. 946414-94-4 and is also known as 5C4, BMS-936558, MDX-1106, and ONO-4538. The preparation and characteristics of nivolumab are described in U.S. Patent No. 8,008,449 and International Patent Publication No. WO 2006/121168, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of nivolumab in various forms of cancer have been described in Wang et al., Cancer Immunol. Res. 2014, 2, 846-56; Page et al., Ann. Rev. Med., 2014, 65, 185-202; and Weber et al., J. Clin. Oncology, 2013, 31, 4311-4318, the contents of which are incorporated herein by reference. The amino acid sequence of nivolumab is presented in Table 18. Nivolumab has intra-heavy chain disulfide bonds at 22-96, 140-196, 254-314, 360-418, 22”-96”, 140”-196”, 254”-314”, and 360”-418”; intra-heavy chain disulfide bonds at 23”-88”, 134”-194”’, 23”’-88”’, and 134”’-194”’; inter-heavy chain disulfide bonds at 127-214”’ and 127”-214”’; inter-heavy chain disulfide bonds at 219-219” and 222-222”; and N-glycosylation sites (H CH2 84.4) at 290” and 290”.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:158 and the light chain provided in SEQ ID NO:159. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO:159, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of nivolumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:160, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:161 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:165, SEQ ID NO:166, and SEQ ID NO:167, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of nivolumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab.

Table 18. Amino acid sequences of PD-1 inhibitors associated with nivolumab.

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at the following doses: about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 240 mg every 2 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 480 mg every 4 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma, with approximately 1 mg/kg of nivolumab administered every 3 weeks on the same day, followed by 3 mg/kg of ipilimumab for 4 consecutive doses, then 240 mg every 2 weeks or 480 mg of nivolumab every 4 weeks.

In some implementations, nivolumab is administered as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks and ipilimumab at approximately 1 mg/kg every 6 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks, plus ipilimumab at 1 mg/kg every 6 weeks and two cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks or 480 mg every 4 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks and ipilimumab at 1 mg/kg every 6 weeks to treat malignant pleural mesothelioma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks and 480 mg of nivolumab every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks to treat pediatric patients <40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 240 mg nivolumab every 2 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 480 mg nivolumab every 4 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab is administered every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 480 mg of nivolumab is administered every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor comprises pembrolizumab (available from Merck Inc. of Kenilworth, NJ, USA under the KEYTRUDA license) or its antigen-binding fragment, conjugate, or variant. Pembrolizumab is designated CAS Registry No. 1374853-91-4 and is also known as lanizumab, MK-3475, and SCH-900475. Pembrolizumab is an immunoglobulin G4 anti-(human protein PDCD1 (programmed cell death 1)) antibody containing a (human-mouse monoclonal heavy chain) disulfide bond and a human-mouse monoclonal light chain dimer structure. The structure of pembrolizumab can also be described as an immunoglobulin G4 anti-(human programmed cell death 1) antibody; containing a humanized mouse monoclonal [228-L-proline (H10-S>P)]γ4 heavy chain (134-218') disulfide bond and a humanized mouse monoclonal κ light chain dimer (226-226”:229-229”) disulfide bond. The properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO 2008/156712 A1, US Patent No. 8,354,509, and US Patent Application Publications Nos. US2010/0266617 A1, US2013/0108651A1, and US2013/0109843 A2, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of pembrolizumab in various forms of cancer are described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert et al., Lancet, 2014, 384, 1109-17; and Thomas et al., Exp. Opin. Biol. Ther., 2014, 14, 1061-1064. The amino acid sequence of pembrolizumab is illustrated in Table 19. Pembrolizumab includes the following disulfide bridges: 22-96, 22”-96”, 23”-92”’, 23”’-92”’, 134-218”, 134”-218”’, 138”-198”, 138”’-198”’, 147-203”, 147”-203”, 226-226”, 229-229”, 261-321”, 261”-321”, 367-425”, and 367”-425”; and the following glycosylation sites. (N): Asn-297 and Asn-297". Pembrolizumab is an IgG4/κ isoform containing a stable S228P mutation in the Fc region; the insertion of this mutation in the IgG4 hinge region prevents the formation of the half-molecule typically observed in IgG4 antibodies. Pembrolizumab is heteroglycosylated at Asn297 in the Fc domain of each heavy chain, resulting in a molecular weight of approximately 149 kDa for the complete antibody. The major glycoform of pembrolizumab is the fucosylated degalactosyl biantennary (GOF) form.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:168 and the light chain provided in SEQ ID NO:169. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of pembrolizumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:170, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:171 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises _{VH and VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises VH and VL regions} , each at least 97% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:175, SEQ ID NO:176, and SEQ ID NO:177, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of PD-1 approved by a regulatory agency, referencing pembrolizumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab.

Table 19. Amino acid sequences of PD-1 inhibitors associated with pembrolizumab.

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat melanoma. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat melanoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat melanoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat NSCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat small cell lung cancer (SCLC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat SCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat SCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat head and neck squamous cell carcinoma (HNSCC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat HNSCC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HNSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, children are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat MSI-H or dMMR cancer. In some implementations, pediatrics are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat MSI-H or dMMR cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient colorectal cancer (dMMR CRC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat MSI-H or dMMR CRC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat gastric cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat gastric cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat esophageal cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat esophageal cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cervical cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cervical cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat hepatocellular carcinoma (HCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are treated with pembrolizumab at approximately 200 mg every 3 weeks for Merkel cell carcinoma (MCC). In some implementations, adults are treated with pembrolizumab at approximately 400 mg every 6 weeks for MCC. In some implementations, children are treated with pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks for MCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat renal cell carcinoma (RCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks and axitinib 5 mg is administered orally twice daily to treat RCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat endometrial cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks, and lenvatinib 20 mg is administered orally once daily for non-MSI-H or dMMR tumors to treat endometrial cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are given pembrolizumab at approximately 200 mg every 3 weeks to treat TMB-H cancer. In some implementations, adults are given pembrolizumab at approximately 400 mg every 6 weeks to treat TMB-H cancer. In some implementations, children are given pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat TMB-H cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cutaneous squamous cell carcinoma (cSCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat triple-negative breast cancer (TNBC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat TNBC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, if the patient or subject is an adult, i.e., for the treatment of an adult indication, an additional dosing regimen of 400 mg every 6 weeks may be used. In some implementations, pembrolizumab administration may be initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration may be initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 clone J43 (catalog number BE0033-2) and RMP1-14 (catalog number BE0146) (Bio XCell, West Lebanon, New Hampshire, USA). Many commercially available anti-PD-1 antibodies are known to those skilled in the art.

In some embodiments, the PD-1 inhibitor is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publications Nos. 2010/0266617A1, 2013/0108651A1, and 2013/0109843A2, the disclosures of which are incorporated herein by reference. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody described in U.S. Patent Nos. 8,287,856, 8,580,247, and 8,168,757 and U.S. Patent Application Publications Nos. 2009/0028857A1, 2010/0285013A1, 2013/0022600A1, and 2011/0008369A1, the teachings of which are hereby incorporated by reference. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in U.S. Patent No. 8,735,553B1, the disclosure of which is incorporated herein by reference. In some embodiments, the PD-1 inhibitor is pilithumab, also known as CT-011, described in U.S. Patent No. 8,686,119, the disclosure of which is incorporated herein by reference.

In some embodiments, the PD-1 inhibitor may be a small molecule or a peptide or peptide derivative, such as those described in U.S. Patent Nos. 8,907,053, 9,096,642, and 9,044,442, and U.S. Patent Application Publication No. US2015/0087581; 1,2,4-oxadiazole compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2015/0073024; cyclic peptide mimics and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042; cyclic compounds and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042. Those described in US2015/0125491; 1,3,4-oxadiazole and 1,3,4-thiadiazole compounds and derivatives, such as those described in International Patent Application Publication WO 2015/033301; peptide-based compounds and derivatives, such as those described in International Patent Application Publications WO 2015/036927 and WO 2015/04490; or peptide-based macrocyclic compounds and derivatives, such as those described in US Patent Application Publication US2014/0294898; the disclosure of each of these patents is hereby incorporated by reference in its entirety. In some embodiments, the PD-1 inhibitor is cemiplimab, which is available from Regeneron Corporation.

In some embodiments, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors, antagonists, or blockers described in more detail below. Regarding PD-L1 and PD-L2 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor may also refer to the compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

In some embodiments, the compositions, processes, and methods described herein include PD-L1 or PD-L2 inhibitors. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2. In some embodiments, the antibody competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selective for PD-L1 because the concentration at which the compound binds to or interacts with PD-L1 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L2 receptor. In some embodiments, the compound binds to the PD-L1 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L2 receptor.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2 because the concentration at which the compound binds to or interacts with PD-L2 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L1 receptor. In some embodiments, the compound binds to the PD-L2 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L1 receptor.

Unbound by any theory, it is believed that tumor cells express PD-L1, and T cells express PD-1. However, tumor cell expression of PD-L1 is not required for the efficacy of PD-1 or PD-L1 inhibitors or blockers. In some embodiments, tumor cells express PD-L1. In other embodiments, tumor cells do not express PD-L1. In some embodiments, the method may include a combination of PD-1 and PD-L1 antibodies (e.g., the PD-1 and PD-L1 antibodies described herein) with TIL. The combination of PD-1 and PD-L1 antibodies with TIL may be administered simultaneously or sequentially.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that binds to human PD-L1 and/or PD-L2 with a KD of about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, or about 30 pM or less.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that _{bind to human PD-L1 and/or PD-L2 at a k-association rate of} about ^7.5 × 10⁵ ¹ /M·s or faster, at about 8 × 10⁵ 1/M·s or _faster , at about 8.5 × 10⁵ ¹ /M·s or faster, at about 9 × ^10⁵ 1/M·s or _faster , at about 9.5 × ^10⁵ 1/M·s or _faster , or at about 1 × ^10⁶ 1/M·s or faster. 1/M·s or faster k- _{associations bind} to human PD-L1 and/or PD-L2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that bind to human PD-L1 or PD-L2 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k-dissociation rate of about 2.1 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.3 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, to human PD-1 with _{a k-dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, and to human PD-1 with a k _{-dissociation rate} of about 2.6 × ^{10⁻⁵ 1/s} or slower. It binds to human PD-1 at a k _{dissociation rate} of 1/s or slower, to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 2.7 × ^10⁻⁵ 1/s or slower, or to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, with an IC50 of about 9 nM or less, with an IC50 of about 8 nM or less, with an IC50 of about 7 nM or less, and with an IC50 of about 6 nM or less. It can either inhibit the binding of human PD-L1 or human PD-L2 to human PD-1, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower, or block human PD-1 or block the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-L1 inhibitor is durvalumab, also known as MEDI4736 (available from Medimmune, LLC (a subsidiary of AstraZeneca plc) in Gaithersburg, Maryland), or its antigen-binding fragment, conjugate, or variant. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013/0034559, the disclosure of which is incorporated herein by reference. The clinical efficacy of durvalumab has been described in Page et al., Ann. Rev. Med., 2014, 65, 185-202; Brahmer et al., J. Clin. Oncol. 2014, 32, 5s (Supplement, Abstract 8021); and McDermott et al., Cancer Treatment Rev., 2014, 40, 1056-64. The preparation and characterization of durvalumab are described in U.S. Patent No. 8,779,108, the disclosure of which is incorporated herein by reference. The amino acid sequence of durvalumab is set forth in Table 20. The durvalumab monoclonal antibody comprises disulfide bonds at 22-96, 22”-96”, 23”-89”’, 23”’-89”’, 135”-195”’, 135”’-195”’, 148”-204”, 148”-204”, 215”-224”, 215”’-224”, 230-230”, 233-233”, 265-325”, 265”-325”, 371-429” and 371”-429”’; and N-glycosylation sites at Asn-301” and Asn-301”.

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:178 and the light chain provided in SEQ ID NO:179. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of the durvalumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:180, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:181 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises VH and _{VL regions} , each at least 99% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:182, SEQ ID NO:183, and SEQ ID NO:184, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:185, SEQ ID NO:186, and SEQ ID NO:187, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of anti-PD-L1 approved by a regulatory agency, referencing durvalumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab.

Table 20. Amino acid sequences of PD-L1 inhibitors associated with durvalumab.

In some embodiments, the PD-L1 inhibitor is avelumab, also known as MSB0010718C (available from MerckKGaA/EMD Serono) or its antigen-binding fragment, conjugate, or variant. The preparation and characterization of avelumab are described in U.S. Patent Application Publication No. US2014/0341917 A1, the disclosure of which is incorporated herein by reference. The amino acid sequence of avelumab is set forth in Table 21. Avirumab contains heavy chain intra-chain disulfide bonds (C23-C104) at 22-96, 147-203, 264-324, 370-428, 22”-96”, 147”-203”, 264”-324”, and 370”-428”; and light chain intra-chain disulfide bonds (C23-C104) at 22’-90”, 138’-197”, 22”’-90”’, and 138”’-197”’; 223- Intra-heavy disulfide bonds between the heavy and light chains at 215' and 223"-215"' (h 5-CL 126); intra-heavy disulfide bonds between the heavy and light chains at 229"-229" and 232"-232" (h11, h 14); N-glycosylation sites at 300" and 300" (H CH2 N84.4); fucoidan-like CHO polysaccharides of the fucoidan complex; and H CHS K2 C-terminal lysine cleavage at 450" and 450".

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:188 and the light chain provided in SEQ ID NO:189. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of avirumab. In some embodiments, the heavy chain variable region (VH) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:190, and the light chain variable region (VL) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:191 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:195, SEQ ID NO:196, and SEQ ID NO:197, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of an anti-PD-L1 antibody approved by a pharmaceutical regulatory agency, referencing avirumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. The anti-PD-L1 antibody may be authorized by a pharmaceutical regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is avelumab.

Table 21. Amino acid sequences of PD-L1 inhibitors associated with avirumab.

In some embodiments, the PD-L1 inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (available from Genentech (a subsidiary of Roche Holding AG) in Basel, Switzerland, under the TECENTRIQ license), or an antigen-binding fragment, conjugate, or variant thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent Application Publications Nos. 2010/0203056A1, 2013/0045200A1, 2013/0045201A1, 2013/0045202A1, or 2014/0065135A1, the disclosure of which is specifically incorporated herein by reference. The preparation and characterization of atezolizumab are described in U.S. Patent No. 8,217,149, the disclosure of which is incorporated herein by reference. The amino acid sequence of atezolizumab is set forth in Table 22. Atezolizumab has intrachain disulfide bonds (C23-C104) at 22-96, 145-201, 262-322, 368-426, 22”-96”, 145”-201”, 262”-322”, and 368”-426”; and at 23”-88”, 134”-194”, 23”’-88”’, and 134”’-194”’. Intra-light chain disulfide bonds (C23-C104); intra-heavy chain disulfide bonds at 221-214' and 221”-214”' (h 5-CL 126); intra-heavy chain disulfide bonds at 227-227” and 230-230” (h 11, h 14); and N-glycosylation sites at 298 and 298' (H CH2 N84.4>A).

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:198 and the light chain provided in SEQ ID NO:199. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of atezolizumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:200, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:201 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, _respectively . In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:204, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:205, SEQ ID NO:206, and SEQ ID NO:207, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the anti-PD-L1 antibody is a biosimilar monoclonal antibody of atezolizumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab.

Table 22. Amino acid sequences of PD-L1 inhibitors associated with atezolizumab.

In some embodiments, the PD-L1 inhibitor includes those antibodies described in U.S. Patent Application Publication No. 2014/0341917 A1, the disclosure of which is incorporated herein by reference. In other embodiments, an antibody competing with any of these antibodies for binding to PD-L1 is also included. In some embodiments, the anti-PD-L1 antibody is MDX-1105, also known as BMS-935559, disclosed in U.S. Patent No. 7,943,743, the disclosure of which is incorporated herein by reference. In some embodiments, the anti-PD-L1 antibody is selected from the anti-PD-L1 antibody disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference.

In some embodiments, the PD-L1 inhibitor is a commercially available monoclonal antibody, such as INVIVOMAB anti-m-PD-L1 clone 10F.9G2 (catalog number BE0101, Bio X Cell, West Lebanon, New Hampshire, USA). In some embodiments, the anti-PD-L1 antibody is a commercially available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). Many commercially available anti-PD-L1 antibodies are known to those skilled in the art.

In some implementations, the PD-L2 inhibitor is a commercially available monoclonal antibody, such as BIOLEGEND24F.10C12 mouse IgG2a, κ isotype (catalog number 329602, Biolegend, San Diego, California), SIGMA anti-PD-L2 antibody (catalog number SAB3500395, Sigma-Aldrich, St. Louis, Missouri), or other commercially available anti-PD-L2 antibodies known to those skilled in the art.

2. Combination with CTLA-4 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, including TIL and one or more CTLA-4 inhibitors.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily and is expressed on the surface of helper T cells. CTLA-4 is a negative regulator of CD28-dependent T cell activation and acts as a checkpoint for adaptive immune responses. Similar to the T cell costimulatory protein CD28, CTLA-4 binds to antigens and presents CD80 and CD86 on cells. CTLA-4 delivers inhibitory signals to T cells, while CD28 delivers stimulatory signals. Human antibodies against human CTLA-4 have been described as immunomodulators for many disease conditions, such as for the treatment or prevention of viral and bacterial infections and for the treatment of cancer (WO 01/14424 and WO 00/37504). Several fully human anti-human CTLA-4 monoclonal antibodies (mAbs) have been investigated in clinical trials for the treatment of various types of solid tumors, including (but not limited to) ipilimumab (MDX-010) and trimemumab (CP-675,206).

In some embodiments, the CTLA-4 inhibitor may be any CTLA-4 inhibitor or CTLA-4 blocker known in the art. In particular, it is one of the CTLA-4 inhibitors or blockers described in more detail below. Regarding CTLA-4 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or bioanalyte. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor may also refer to a small molecule compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

CTLA-4 inhibitors suitable for the methods of the present invention include (but are not limited to) anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (ipilimumab), trimemumab, anti-CD28 antibodies, anti-CTLA-4 adenettin, anti-CTLA-4 domain antibodies, and single-chain anti-CTLs. The following are listed: A-4 fragment, heavy chain anti-CTLA-4 fragment, light chain anti-CTLA-4 fragment, CTLA-4 inhibitor activating the co-stimulatory pathway, antibody disclosed in PCT Publication No. WO 2001/014424, antibody disclosed in PCT Publication No. WO 2004/035607, antibody disclosed in U.S. Publication No. 2005/0201994, and antibody disclosed in European Patent No. EP 1212422 B1, the disclosures of each of which are incorporated herein by reference. Other CTLA-4 antibodies are described in U.S. Patents 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publications WO 01/14424 and WO 00/37504; and U.S. Publications 2002/0039581 and 2002/086014, the contents of each of which are incorporated herein by reference. Other anti-CTLA-4 antibodies that may be used in the methods of this invention include, for example, antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (Antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998); and U.S. Patents 5,977,318, 6,682,736, 7,109,003 and 7,132,281, the disclosure of each of which is incorporated herein by reference.

Other CTLA-4 inhibitors include (but are not limited to) the following: those that, by means of activation, disrupt the ability of CD28 antigen to bind to its homologous ligand, inhibit the ability of CTLA-4 to bind to its homologous ligand, enhance T cell responses via the co-stimulatory pathway, disrupt the ability of B7 to bind to CD28 and/or CTLA-4, disrupt the ability of B7 to activate the co-stimulatory pathway, disrupt the ability of CD80 to bind to CD28 and/or CTLA-4, disrupt the ability of CD80 to activate the co-stimulatory pathway, disrupt the ability of CD86 to bind to CD28 and/or CTLA-4, disrupt the ability of CD86 to activate the co-stimulatory pathway, and any inhibitor that disrupts the co-stimulatory pathway. This must include: small molecule inhibitors of CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antibodies against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antisense molecules against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; adenettin against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; RNAi inhibitors (single-stranded and double-stranded) against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; and other CTLA-4 inhibitors.

In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 at a K d , which is about 10  -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, for example, between 10 -13 M and 10 -16 M, or within any range with any two of the foregoing values as endpoints. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is no more than 10 times that of the Kd of ipilimumab. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is substantially the same as or less than that of ipilimumab (e.g., up to 10 times or up to 100 times lower). In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is no more than 10-fold higher than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively. In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is approximately the same as or less than (e.g., up to 10-fold or up to 100-fold lower) than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively.

In some embodiments, a CTLA-4 inhibitor is used in an amount sufficient to inhibit CTLA-4 expression and/or reduce the biological activity of CTLA-4 by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100%. In some embodiments, a CTLA-4 pathway inhibitor is used in an amount sufficient to reduce the biological activity of CTLA-4 by reducing the binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100% relative to a suitable control. In the context of assessing or quantifying the effect of an agent of interest, a suitable control is typically a biological system (e.g., cells or a subject) that has not been exposed to the agent of interest (e.g., a CTLA-4 pathway inhibitor) or has been treated with the agent (or has been exposed to or treated with a negligible amount). In some embodiments, the biological system may serve as its own control, for example, by assessing the biological system before exposure to or treatment with the agent and comparing its status after the start or end of exposure or treatment. In some embodiments, a historical control may be used.

In some embodiments, the CTLA-4 inhibitor is ipilimumab (available from Bristol-Myers Squibb at Yervoy) or its biosimilars, antigen-binding fragments, conjugates, or variants. As known in the art, ipilimumab refers to an anti-CTLA-4 antibody, a fully human IgG 1κ antibody derived from transgenic mice having human genes encoding both heavy and light chains to produce a functional human lineage. Ipilimumab is also mentioned in its CAS Registry No. 477202-00-9 and in PCT Publication No. WO 01/14424, which is incorporated herein by reference in its entirety for all purposes. It is disclosed in the form of Antibody 10DI. Specifically, ipilimumab contains a light chain variable region and a heavy chain variable region (having a light chain variable region comprising SEQ ID NO: 211 and a heavy chain variable region comprising SEQ ID NO: 210). Pharmaceutical compositions of ipilimumab comprise all pharmaceutically acceptable compositions containing ipilimumab and one or more diluents, mediators, or excipients. Examples of pharmaceutical compositions containing ipilimumab are described in International Patent Application Publication No. WO 2007/67959. Ipilimumab can be administered intravenously (IV).

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:208 and the light chain provided in SEQ ID NO:209. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of ipilimumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:210, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:211 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:215, SEQ ID NO:216, and SEQ ID NO:217, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to ipilimumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of ipilimumab is illustrated in Table 23. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab.

Table 23. Amino acid sequence of ipilimumab.

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, ipilimumab administration may be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, ipilimumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab is administered at approximately mg/kg every 3 weeks for up to 4 consecutive doses to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered as adjuvant therapy for melanoma. In some implementations, ipilimumab is administered at approximately 10 mg/kg every 3 weeks for four doses, followed by 10 mg/kg every 12 weeks for up to 3 years as adjuvant therapy for melanoma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat advanced renal cell carcinoma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 3 weeks, followed by nivolumab at 3 mg/kg on the same day, for a total of 4 doses to treat advanced renal cell carcinoma. In some implementations, after completing the combined 4-dose regimen, nivolumab can be administered as a single agent against advanced renal cell carcinoma and/or renal cell carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, ipilimumab is administered intravenously at approximately 1 mg/kg every 30 minutes over 3 weeks, followed immediately by nivolumab intravenously at 3 mg/kg every 30 minutes on the same day, for a total of 4 doses to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, after completing 4 doses of the composition, nivolumab is administered as a single agent for microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, as recommended according to standard dosing regimens. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also begin 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat hepatocellular carcinoma. In some implementations, ipilimumab is administered intravenously at approximately 3 mg/kg every 3 weeks over 30 minutes, followed immediately by nivolumab intravenously at 1 mg/kg over 30 minutes on the same day, for a total of four doses to treat hepatocellular carcinoma. In some implementations, after completing the combined four-dose regimen, nivolumab is administered as a single agent against hepatocellular carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, followed by nivolumab at 3 mg/kg every 2 weeks to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, plus 360 mg nivolumab every 3 weeks for 2 cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat malignant pleural mesothelioma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks and nivolumab at 360 mg every 3 weeks to treat malignant pleural mesothelioma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

Trimelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has CAS number 745013-59-6. Trimelimumab is disclosed in U.S. Patent No. 6,682,736 (incorporated herein by reference) as antibody 11.2.1. The amino acid sequences of the heavy and light chains of trimemumab are described in SEQ IND NO: 218 and 219, respectively. Trimelimumab has been studied in clinical trials for the treatment of various tumors, including melanoma and breast cancer; wherein it is administered intravenously in single or multiple doses at a dose range between 0.01 and 15 mg/kg every 4 or 12 weeks. In the embodiments provided by the present invention, trimemumab is administered topically, particularly intradermally or subcutaneously. The effective amount of trimemumab administered intradermally or subcutaneously is typically in the range of 5-200 mg/dose per person. In some implementations, the effective dose of trimemumab is in the range of 10-150 mg/dose per person. In some specific implementations, the effective dose of trimemumab is approximately 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175 or 200 mg/dose per person.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:218 and the light chain provided in SEQ ID NO:219. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of trimexumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:220, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:221 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in _SEQ ID NO:220 and _SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:225, SEQ ID NO:226, and SEQ ID NO:227, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a biosimilar monoclonal antibody of anti-CTLA-4 approved by a regulatory agency, referencing trimemumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of trimemumab is illustrated in Table 24. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab.

Table 24. Amino acid sequence of trimemumab.

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, wherein trimemumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, trimemumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, trimemumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is zeflimab from Agenus or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Zeflimab is a fully human monoclonal antibody. Zeflimab is designated Chemical Abstracts Service (CAS) Registry No. 2148321-69-9 and is also known as AGEN1884. The preparation and characterization of zeflimab are described in U.S. Patent No. 10,144,779 and U.S. Patent Application Publication No. US2020/0024350A1, the disclosures of which are incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:228 and the light chain provided in SEQ ID NO:229. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of zeflimab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:230, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:231 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:231, SEQ ID NO:233, and SEQ ID NO:234, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:235, SEQ ID NO:236, and SEQ ID NO:237, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to zeflimab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of zeflimab is illustrated in Table 25. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab.

Table 25. Amino acid sequence of zefrizinumab.

Other examples of anti-CTLA-4 antibodies include (but are not limited to): AAGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659, and ADG116, which are known to those skilled in the art.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent disclosures: US2019/0048096 A1; US2020/0223907; US2019/0201334; US2019/0201334; US2005/0201994; EP 1212422B1; WO 2018/204760; WO 2018/204760; WO 2001/014424; WO2 WO 2004/035607; WO 2003/086459; WO 2012/120125; WO 2000/037504; WO 2009/100140; WO2006/09649; WO2005092380; WO 2007/123737; WO 2006/029219; WO 2010/0979597; WO2006/12168; and WO1997020574, each of which is incorporated herein by reference. Other CTLA-4 antibodies are described in the following: U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227 and 6,984,720; PCT Publications Nos. WO 01/14424 and WO 00/37504; and U.S. Publications Nos. 2002/0039581 and 2002/086014; and/or U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281, each of which is incorporated herein by reference. In some embodiments, the anti-CTLA-4 antibody is, for example, one of the antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 10067-10071 (1998); Camacho et al., J. Clin. Oncol., 2004, 22, 145 (Abstract No. 2505 (2004) (Antibody CP-675206); or Mokyr et al., Cancer Res., 1998, 58, 5301-5304 (1998), each of which is incorporated herein by reference.

In some implementations, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996/040915 (incorporated herein by reference).

In some implementations, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, anti-CTLA-4 RNAi molecules may be in the form described in: PCT Publications WO 1999/032619 and WO 2001/029058; U.S. Publications 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, and 2005/01009. Patent Nos. 13, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443; and/or U.S. Patent Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in European Patent No. 1309726 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in U.S. Patent Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In some embodiments, the CTLA-4 inhibitor is an aptamer described in International Patent Application Publication No. WO2004/081021 (incorporated herein by reference).

In other embodiments, the anti-CTLA-4 RNAi molecule of the present invention is the RNA molecule described in U.S. Patent Nos. 5,898,031, 6,107,094, 7,432,249 and 7,432,250 and European Application No. EP 0928290 (incorporated herein by reference).

3. Preconditioning of lymphocyte depletion in patients

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the present disclosure. In some embodiments, the present invention includes a TIL population for treating cancer in patients pretreated with non-myeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/ ^m² /day for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion (day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 (aldeleukin, available from PROLEUKIN) every 8 hours at 720,000 IU/kg to achieve physiological tolerance. In some implementations, TIL groups are used in combination with IL-2 to treat cancer, wherein IL-2 is administered after the TIL group.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system ('cytokine pool') prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as "immunosuppressive modulation") in a patient prior to the introduction of the TIL of the present invention.

Generally, lymphocyte depletion is achieved by administration of fludarabine or cyclophosphamide (the active form of which is called equiphosphamide) and combinations thereof. Such methods are described in Gassner et al., Cancer Immunol. Immunother. 2011, 60, 75-85; Muranski et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-5239; and Dudley et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL. In some embodiments, fludarabine is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, fludarabine is administered at doses of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2–7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4–5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4–5 days.

In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 1 μg/mL. In some embodiments, cyclophosphamide is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, cyclophosphamide is administered at doses of 100 mg/m²/day, 150 mg/m²/day, 175 mg/m²/day, 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day, 275 mg/m²/day, or 300 mg/m²/day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.e., i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2–7 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4-5 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4 days.

In some implementations, lymphocyte depletion is achieved by administering fludarabine and cyclophosphamide together to the patient. In some implementations, fludarabine is administered intravenously at 25 mg/m²/day for 4 days, and cyclophosphamide is administered intravenously at 250 mg/m²/day.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days and fludarabine at a dose of 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered for the first two days and lymphocyte depletion is achieved over a total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 15 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by fludarabine at a dose of 25 mg/m²/day for three days.

In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, mesna is administered at a dose of 15 mg/kg. In some embodiments, mesna is infused, and if continuous infusion is initiated over 24 hours, with the respective cyclophosphamide dose starting, mesna may be infused with cyclophosphamide over approximately 2 hours (days -5 and/or -4), and then infused at a rate of 3 mg/kg/hour for the remaining 22 hours.

In some implementations, lymphocyte depletion includes the following steps: starting the day after administering the third TIL group to the patient, treating the patient with an IL-2 regimen.

In some implementations, lymphocyte depletion includes the following steps: starting on the day the third TIL group is administered to the patient, the patient is treated with an IL-2 regimen.

In some embodiments, lymphocyte depletion comprises a 5-day preconditioning treatment. In some embodiments, the days are indicated as day-5 to day-1, or day 0 to day 4. In some embodiments, the regimen comprises cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises 60 mg/kg intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, the regimen also comprises fludarabine. In some embodiments, the regimen also comprises intravenous fludarabine. In some embodiments, the regimen also comprises 25 mg/ ^m² intravenous fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine at a dose of 25 mg/ ^m² on days -5 and ^-1 (i.e., days 0 to 4).

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for one day.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 26.

Table 26. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 27.

Table 27. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 28.

Table 28. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 29.

Table 29. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day			X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 30.

Table 30. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 31.

Table 31. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 32.

Table 32. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 33.

Table 33. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day			X	X	X
TIL infusion						X

In some embodiments, the TIL infusion used in conjunction with the above-described myeloablative lymphocyte depletion regimens may be any TIL composition described herein, as well as an IL-2 addition regimen and administration of combination therapies as described herein (e.g., PD-1 and PD-L1 inhibitors).

4. IL-2 scheme

In some implementations, the IL-2 regimen includes a high-dose IL-2 regimen comprising alephrine or a biosimilar or variant thereof, administered intravenously starting the day after the effective fraction of treatment in the therapeutic TIL population. Alephrine or a biosimilar or variant thereof is administered as a 15-minute bolus intravenous infusion every eight hours at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient weight) until tolerated, for a maximum of 14 doses. After a 9-day rest period, this course can be repeated for another 14 doses, for a maximum total of 28 doses. In some implementations, IL-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In some implementations, IL-2 is administered at a maximum dose of up to 6 doses.

In some implementations, the IL-2 regimen comprises a tapering IL-2 regimen. Tapering IL-2 regimens have been described in O'Day et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In some implementations, tapering IL-2 therapy comprises an intravenous administration of 18 × ^10⁶ IU/ ^m² over 6 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 12 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 24 hours, followed by an intravenous administration of 4.5 × ^10⁶ IU/ ^m² of adeleukin or a biosimilar or variant thereof over 72 hours. This treatment cycle may be repeated every 28 days for a maximum of four cycles. In some implementations, the decreasing IL-2 regimen includes 18,000,000 IU/ ^m² on day 1, 9,000,000 IU/ ^m² on day 2, and 4,500,000 IU/ ^m² on days 3 and 4.

In some implementations, the IL-2 regimen includes a low-dose IL-2 regimen. Any low-dose IL-2 regimen known in the art may be used, including those described in Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann et al., Lancet Diabetes Endocrinol. 2013, 1, 295-305; and Rosenzwaig et al., Ann. Rheum. Dis. 2019, 78, 209-217, the disclosures of which are incorporated herein by reference. In some implementations, a low-dose IL-2 regimen comprises 18 × ^10⁶ IU/ ^m² of interleukin or its biosimilar or variant every 24 hours, administered as a continuous infusion for 5 days; followed by a 2–6 day period without IL-2 therapy; optionally followed by intravenous administration of interleukin or its biosimilar or variant at 18 × ^10⁶ IU/ ^m² every 24 hours for another 5 days; optionally followed by a 3-week period without IL-2 therapy, after which other cycles may be administered.

In some implementations, IL-2 is administered at a maximum dose of up to 6 doses. In some implementations, high-dose IL-2 regimens are modified for pediatric use. In some implementations, interleukin is administered at a dose of 600,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 400,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 300,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 200,000 IU/kg every 8-12 hours, for a maximum of 6 doses. In some implementations, interleukin is administered at a dose of 100,000 IU/kg every 8-12 hours, for a maximum of 6 doses.

In some embodiments, the IL-2 regimen comprises administering PEGylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bepedipine interleukin or a fragment, variant, or bioanalyte thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen comprises administering THOR-707 or a fragment, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen includes administration of Nevorin Alpha or a fragment, variant, or biosimilar thereof following TIL administration. In some implementations, Nevorin Alpha is administered to the patient at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 protocol includes administering an IL-2 fragment grafted onto the antibody backbone. In some embodiments, the IL-2 protocol includes administering an antibody cytokine graft protein that binds to a low-affinity IL-2 receptor. In some embodiments, the antibody cytokine graft protein includes a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof grafted into the CDR of _VH or _VL , wherein the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof, which is transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody or a fragment thereof, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days, wherein the antibody comprises a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

In some implementations, the serum half-life of the antibody cytokine transplanted protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to interleukin or comparable molecules).

In some embodiments, the TIL infusion used in conjunction with the aforementioned embodiments of the myeloablative lymphocyte depletion regimen may be any TIL composition described herein and may also include MIL and PBL infusions in lieu of TIL infusion, as well as the addition of an IL-2 regimen and administration of combination therapies as described herein (e.g., PD-1 and/or PD-L1 inhibitors and/or CTLA-4 inhibitors).

Implementation Plan

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating the fourth TIL population takes about 9 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 bead agonist or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about one day.

In some implementations, the process of cultivating the fourth TIL population takes about 2 days.

In some implementations, the process of cultivating a fourth TIL population takes about 3 days.

In some implementations, the process of cultivating the fourth TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing a third TIL population takes about 8 days.

In some implementations, the process of culturing a third TIL population takes approximately 9 days.

In some implementations, the process of culturing the third TIL population takes about 8-9 days.

In some implementations, the process of cultivating a third TIL population takes about 10 days.

In some implementations, the process of culturing a third TIL population takes approximately 8-10 days.

In some implementations, the process of culturing a third TIL population takes approximately 11 days.

In some implementations, the process of culturing a third TIL population takes approximately 12 days.

In some implementations, the process of cultivating a third TIL population takes approximately 13 days.

In some implementations, the process of culturing a third TIL population takes approximately 14 days.

In some implementations, the process of cultivating a third TIL population takes about 15 days.

In some implementations, the step of culturing a third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about one day.

In some implementations, the process of cultivating a third TIL population takes about 2 days.

In some implementations, the process of cultivating a third TIL population takes about 3 days.

In some implementations, the process of cultivating a third TIL population takes about 4 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) Culture the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) A fourth population of TILs was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately nine days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a third cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a fourth culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the step of culturing a third or fourth TIL population takes about one day.

In some implementations, the process of culturing a third or fourth TIL population takes about 2 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 3 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 4 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 5 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 6 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, during the step of culturing a first TIL population in a first culture medium, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in the first culture medium.

In some embodiments, during the step of culturing a second TIL population in a second culture medium, the second culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in a second culture medium.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of a first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the initiation of the first amplification takes approximately one day.

In some implementations, the initiation of the first amplification takes approximately 2 days.

In some implementations, the initiation of the first amplification takes approximately 3 days.

In some implementations, the initiation of the first amplification takes approximately 4 days.

In some implementations, the initiation of the first amplification takes approximately 5 days.

In some implementations, the initiation of the first amplification takes approximately 6 days.

In some implementations, the initiation of the first amplification takes approximately 7 days.

In some implementations, the initiation of the first amplification takes approximately 8 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the rapid second amplification takes approximately 9 days.

In some implementations, the rapid second amplification takes approximately 10 days.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, all steps are completed within a period of approximately 16 days.

In some implementations, all steps are completed within a period of approximately 17 days.

In some implementations, all steps are completed within a period of approximately 18 days.

In some implementations, all steps are completed within a period of approximately 18-22 days.

In some implementations, all steps are completed within a period of approximately 19 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 21 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, the first culture medium contains APC.

In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

In some embodiments, the first culture medium contains OKT-3.

In some implementations, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies during the initial amplification step.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digesting tumor tissue samples in an enzyme medium to produce tumor digests;

(c) The tumor digest is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (f), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the first amplification takes about 3 days.

In some implementations, the first amplification takes about 4 days.

In some implementations, the first amplification takes about 5 days.

In some implementations, the first amplification takes about 6 days.

In some implementations, the first amplification takes about 7 days.

In some implementations, the first amplification takes about 8 days.

In some implementations, the first amplification takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes about 5 days.

In some implementations, the second amplification takes about 6 days.

In some implementations, the second amplification takes about 7 days.

In some implementations, the second amplification takes approximately 8 days.

In some implementations, the second amplification takes approximately 9 days.

In some implementations, the second amplification takes about 8-9 days.

In some implementations, the second amplification takes about 10 days.

In some implementations, the second amplification takes about 8-10 days.

In some implementations, the second amplification takes approximately 11 days.

In some implementations, the second amplification takes approximately 12 days.

In some implementations, the second amplification takes approximately 13 days.

In some implementations, the second amplification takes approximately 14 days.

In some implementations, the second amplification takes about 15 days.

In some implementations, the second amplification is performed through the following steps:

(i) The first period of culturing the fourth TIL population in the second culture medium for about 5 days.

(ii) The culture from step (i) is subdivided into multiple subcultures, each of which is transferred to a separate sealed container providing a third breathable surface and cultured for a second period of approximately 4 or 5 days in a third medium containing IL-2, wherein the transition from step (i) to step (ii) is performed without opening the system, and

(iii) Multiple subcultures are merged to produce a fifth TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, all steps are completed within a period of approximately 25 days.

In some implementations, all steps are completed within a period of approximately 26 days.

In some implementations, all steps are completed within a period of approximately 27 days.

In some implementations, all steps are completed within a period of approximately 28 days.

In some implementations, all steps are completed within a period of approximately 29 days.

In some implementations, all steps are completed within a period of approximately 30 days.

In some implementations, all steps are completed within a period of approximately 31 days.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Digesting a first TIL population from tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a patient or subject in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, this document provides expanded numbers of tumor-infiltrating lymphocytes (TILs) or therapeutic TIL populations produced by the methods described herein.

In some embodiments, this document provides pharmaceutical compositions comprising an amplified number of TILs or a therapeutic population of TILs as described herein and a pharmaceutically acceptable carrier.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of an expanded number of TILs or a therapeutically active population of TILs as described herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor excised from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor digest to a closed system and performing a first amplification to produce a second TIL population by culturing the first TIL population in a first cell culture medium containing IL-2, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting PD-1 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CISH expression.

In some implementations, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting LAG-3 expression and a TALEN system for inhibiting TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is approximately 2.3 × 10¹⁰ to approximately 13.7 × 10¹⁰ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of TIL cells in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the administration of TIL cells to the patient in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Example

The embodiments covered herein are now described with reference to the following examples. These examples are provided for illustrative purposes only and this disclosure should in no way be construed as limiting to these examples, but rather as covering any and all variations that become apparent from the teachings provided herein.

Example 1: Preparation of culture medium for pre-REP and REP processes

This embodiment describes a procedure for preparing a tissue culture medium suitable for cultures involving tumor-infiltrating lymphocytes (TILs) derived from various solid tumors. This culture medium can be used to prepare any of the TILs described in this application and other embodiments.

Prepare CM1 . Remove the following reagents from the freezer and warm them in a 37°C water bath: (RPMI 1640, human AB serum, 200 mM L-glutamine). Prepare CM1 medium by adding each component to the top of a 0.2 μm filter unit suitable for the desired filtration volume, according to Table 34 below. Store at 4°C.

Table 34. Preparation of CM1

On the day of use, preheat the required amount of CM1 in a 37°C water bath and add 6000 IU/mL IL-2.

Additional information may be added as needed, based on Table 35.

Table 35. Additional information for CM1 as needed.

Preparation of CM2

Remove the prepared CM1 from the refrigerator or prepare fresh CM1. Remove AIM- from the refrigerator and prepare the required amount of CM2 by mixing the prepared CM1 with an equal volume of AIM- in a sterile culture medium bottle. On the day of use, add 3000 IU/mL IL-2 to the CM2 culture medium. On the day of use, prepare a sufficient amount of CM2 with 3000 IU/mL IL-2. Label the CM2 culture medium bottle with the name, the initials of the preparer's name, the filtration/preparation date, and the two-week expiry date, and store at 4°C until needed for tissue culture.

Preparation of CM3

Prepare CM3 on the day it is needed. CM3 is the same as AIM-medium, but supplemented with 3000 IU/mL IL-2 on the day of use. Prepare the amount of CM3 needed for the experiment by directly adding the IL-2 stock solution to the AIM-V bottle or bag. Mix thoroughly by gentle shaking. Immediately after adding to AIM-V, label the bottle “3000 IU/mL IL-2”. If there is excess CM3, store it in a bottle at 4°C, labeling it with the medium name, the preparer's initials, the date of preparation, and its expiration date (7 days after preparation). After storing at 4°C for 7 days, discard the medium supplemented with IL-2.

Preparation of CM4

CM4 is the same as CM3, but with an additional 2 mM GlutaMAX ^™ (final concentration). Add 10 mL of 200 mM GlutaMAX ^™ per 1 L of CM3. Prepare the required amount of CM4 by directly adding IL-2 and GlutaMAX ^™ stock solution to the AIM-V bottle or bag. Mix thoroughly with gentle shaking. Immediately after adding to the AIM-V, label the bottle “3000 IL/mL IL-2 and GlutaMAX”. If excess CM4 exists, store it in the bottle at 4°C, labeling it with the culture medium name, “GlutaMAX”, and its expiration date (7 days after preparation). Discard the culture medium supplemented with IL-2 after storage at 4°C for more than 7 days.

Example 2: Use of a mixture of IL-2, IL-15 and IL-21 cytokines

This embodiment describes the use of IL-2, IL-15, and IL-21 cytokines, which act as additional T cell growth factors, in combination with the TIL process of any embodiment herein.

Using the procedures described herein, at the start of culture, TILs were observed to grow from tumors in one experimental group in the presence of IL-2 and in another group in the presence of a combination of IL-2, IL-15, and IL-21, which substituted for IL-2. At the completion of pre-REP, the amplification, phenotype, function (CD107a+ and IFN-γ), and TCR Vβ lineage of the cultures were assessed. IL-15 and IL-21 are described elsewhere in this paper and in Santegoets et al., J. Transl. Med., 2013, 11, 37.

The results showed enhanced TIL amplification (>20%) in CD4 ⁺ and CD8 ⁺ cells under IL-2, IL-15, and IL-21 treatments compared to IL-2-only conditions. Significant skewness towards a CD8 ⁺ population with a skewed TCR Vβ lineage was observed in TILs obtained from IL-2, IL-15, and IL-21-treated cultures compared to IL-2-only cultures. IFN-γ and CD107a levels were increased in TILs treated with IL-2, IL-15, and IL-21 compared to IL-2-only TILs.

Example 3: Quantification of individual batches of γ-irradiated peripheral mononuclear cells

This embodiment describes a simplified procedure for quantifying individual batches of γ-irradiated peripheral monocytes (PBMCs, also known as monocytes or MNCs) used as allogeneic feeder cells in the exemplary methods described herein.

Each batch of irradiated MNC feeder cells was prepared from individual donors. Each batch or donor was individually screened based on its ability to amplify TILs in the REP in the presence of purified anti-CD3 (clonal OKT3) antibody and interleukin-2 (IL-2). Furthermore, each batch of feeder cells was tested without the addition of TILs to verify that the received gamma irradiation dose was sufficient to prevent replication.

TIL regeneration requires γ-irradiated, growth-arrested MNC feeder cells. Membrane receptors on the feeder MNCs bind to anti-CD3 (clonal OKT3) antibodies and cross-link with TILs in the REP culture flask, stimulating TIL expansion. Feeder cell batches are prepared by leukocyte removal from whole blood obtained from individual donors. The leukocyte removal product is centrifuged, washed, irradiated, and cryopreserved under GMP conditions on a Ficoll-Hypaque apparatus.

Importantly, live feeder cells should not be infused into patients receiving TIL therapy, as this can cause graft-versus-host disease (GVHD). Consequently, feeder cell growth is inhibited by gamma irradiation, leading to double-strand DNA breaks and loss of MNC cell viability upon reculture.

Feeder cell batches were evaluated according to two criteria: (1) their ability to amplify TILs by >100-fold in co-culture, and (2) their insufficient replication capacity.

Feeder cell batches were tested in a mini-REP format using two primary pre-REP TIL lines grown in vertical T25 tissue culture flasks. Feeder cell batches were tested against two distinct TIL lines, each with a unique ability to proliferate in response to activation within the REP. As a control, a number of previously demonstrated irradiated MNC feeder cells meeting the above criteria were processed with the test batches.

A stock solution of the same pre-REP TIL line is available to test all conditions and all feeder cell batches, ensuring that all batches tested in a single experiment undergo equivalent testing.

For each batch of feeder cells tested, a total of six T25 culture flasks were used: pre-REP TIL line #1 (2 flasks); pre-REP TIL line #2 (2 flasks); and feeder cell control (2 flasks). Flasks containing TIL lines #1 and #2 were used to evaluate the feeder cell batch's ability to amplify TILs. The feeder cell control flasks were used to evaluate insufficient replication capacity in the feeder cell batch.

A. Experimental Protocol

On day -2/3, thaw the TIL culture . Prepare CM2 medium and warm it in a 37°C water bath. Prepare 40 mL of CM2 supplemented with 3000 IU/mL IL-2. Keep warm until used. Place 20 mL of pre-warmed CM2 (without IL-2) into each of two 50 mL conical tubes labeled with the name of the TIL culture used. Remove the two designated pre-REP TIL cultures from the LN2 reservoir and transfer the vials to the tissue culture room. Thaw the vials by placing them in a sealed zip-lock storage bag in a 37°C water bath until a small amount of ice remains.

Using a sterile pipette, immediately transfer the contents of each vial to 20 mL of CM2 in a prepared, labeled 50 mL conical tube. Make up to 40 mL with IL-2-free CM2 to wash the cells and centrifuge at 400×CF for 5 minutes. Aspirate the supernatant and resuspend the cells in 5 mL of warm CM2 supplemented with 3000 IU/mL IL-2.

Take two aliquots (20 μL each) for cell counting using an automated cell counter. Record the counts. During counting, place a 50 mL conical tube containing TIL cells in a humidified 37°C, 5% _CO2 incubator with the cap loosened to allow gas exchange. Determine the cell concentration and dilute the TIL to 1 × ^10⁶ cells/mL in CM₂ supplemented with 3000 IU/mL IL-2.

In a humidified 37°C incubator, culture at 2 ml/well in multiple wells of a 24-well tissue culture plate as needed until day 0 of the micro-REP. Culture different TIL lines in separate 24-well tissue culture plates to avoid confusion and potential cross-contamination.

Day 0, start the micro-REP . Prepare sufficient CM2 medium for the number of feeder cell batches to be tested. (For example, prepare 800 mL of CM2 medium for testing 4 feeder cell batches at once). Divide a portion of the CM2 prepared above into equal parts and supplement it with 3000 IU/mL IL-2 for cell culture. (For example, prepare 500 mL of CM2 medium with 3000 IU/mL IL-2 for testing 4 feeder cell batches at once).

To prevent cross-contamination, the 24-well plates containing TIL cultures were removed from the incubator and transferred to the BSC.

Using sterile pipettes or 100-1000 μL pipettes and tips, remove approximately 1 mL of culture medium from each well to be used and place it into an unused well of a 24-well tissue culture plate.

Using fresh, sterile pipettes or 100-1000 μL pipettes and tips, mix the remaining culture medium with the TIL in the wells to resuspend the cells, and then transfer the cell suspension to 50 mL conical tubes labeled with the TIL batch name and record the volume.

Wash each well with the reserved culture medium and transfer the volume to the same 50 mL conical tube. Rotate the cells at 400 × CF to collect the cell pellet. Drain the culture supernatant and resuspend the cell pellet in 2–5 mL of CM2 medium containing 3000 IU/mL IL-2. The volume to be used is based on the number of wells collected and the size of the pellet; that is, the volume should be sufficient to ensure a concentration >1.3 × ^10⁶ cells/mL.

Using a serum pipette, thoroughly mix the cell suspension and record the volume. Take 200 μL for cell counting using an automated cell counter. During counting, place a 50 mL conical tube containing TIL cells in a humidified 5% _CO2 incubator at 37°C with the cap loosened to allow gas exchange. Record the count.

Remove the 50 mL conical tube containing TIL cells from the incubator and resuspend the cells at a concentration of 1.3 × ^10⁶ cells/mL in warm CM2 supplemented with 3000 IU/mL IL-2. Place the 50 mL conical tube back into the incubator and loosen the cap.

For the second TIL system, repeat the above steps.

Before plating TIL into the T25 culture flasks used for experiments, dilute TIL 1:10 to a final concentration of 1.3 × ^10⁵ cells/mL as shown below.

Prepare a working solution of pure CD3 (OKT3) from MACS GMP . Remove the OKT3 stock solution (1 mg/mL) from the 4°C freezer and place it in BSC. Use OKT3 at a final concentration of 30 ng/mL in the micro-REP medium.

In each T25 culture flask used in the experiment, 600 ng OKT3 is required per 20 mL; this is equivalent to 60 μL of 10 μg/mL solution per 20 mL, or 360 μL for all 6 culture flasks tested for each feeder cell batch.

For each feeder cell batch tested, prepare 400 μL of a 1:100 dilution of 1 mg/mL OKT3 for a working concentration of 10 μg/mL (e.g., for a single test of 4 feeder cell batches, prepare 1600 μL of a 1:100 dilution of 1 mg/mL OKT3: 16 μL of 1 mg/mL OKT3 + 1.584 mL of CM2 medium with 3000 IU/mL IL-2).

Prepare T25 culture flasks . Label each culture flask and fill it with CM2 medium before preparing feeder cells. Place the flasks in a humidified 5% _CO2 incubator at 37°C to keep the medium warm while awaiting the addition of the remaining components. After preparing feeder cells, add the components to the CM2 in each culture flask.

Other information is provided in Table 36.

Table 36. Solution Information.

Prepare feeder cells . For this protocol, each batch tested requires at least 78 × ^10⁶ feeder cells. Each 1 mL vial frozen by SDBB has 100 × ^10⁶ viable cells at freezing. Assuming a 50% recovery rate after thawing from a liquid _N₂ reservoir, it is recommended to thaw at least two 1 mL vials of feeder cells per batch, thus using an estimated 100 × ^10⁶ viable cells per REP. Alternatively, if supplied in 1.8 mL vials, a single vial may provide sufficient feeder cells.

Before thawing the feeder cells, pre-warm approximately 50 mL of IL-2-free CM2 for each feeder cell batch to be tested. Remove the designated feeder cell batch vial from the LN2 reservoir, place it in a zip-lock storage bag, and keep it on ice. Thaw the vial in the sealed zip-lock storage bag by immersion in a 37°C water bath. Remove the vial from the zip-lock bag, spray or wipe it with 70% EtOH, and transfer it to a BSC.

Using a pipette, immediately transfer the contents of the feeder cell vial to 30 mL of warm CM2 in a 50 mL conical tube. Wash the vial with a small volume of CM2 to remove any residual cells and centrifuge at 400×CF for 5 min. Aspirate the supernatant and resuspend the cells in 4 mL of warm CM2 with 3000 IU/mL IL-2. Take 200 μL for cell counting using an automated cell counter. Record the count.

Cells were resuspended at 1.3 × ^10⁷ cells/mL in warm CM2 with 3000 IU/mL IL-2. TIL cells were diluted from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL.

Set up the co-culture . Dilute TIL cells from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL. Add 4.5 mL of CM2 medium to a 15 mL conical tube. Remove the TIL cells from the incubator and resuspend them thoroughly using a 10 mL serum pipette. Take 0.5 mL of the 1.3 × ^10⁶ cells/mL TIL suspension and add it to 4.5 mL of medium in a 15 mL conical tube. Return the TIL stock solution vial to the incubator. Mix thoroughly. Repeat the above steps for the second TIL line.

Transfer the culture flasks containing pre-warmed medium for a single feeder cell batch from the incubator to the BSC. Mix the feeder cells by pipetting several times up and down with a 1 mL pipette tip, and transfer 1 mL (1.3 × ^10⁷ cells) to each culture flask containing the feeder cell batch. Add 60 μL of OKT3 working stock solution (10 μg/mL) to each culture flask. Return the two control flasks to the incubator.

Transfer 1 mL (1.3 × ^10⁵ ) of each TIL batch to a correspondingly labeled T25 culture flask. Place the flask back into the incubator and incubate upright. Begin the intervention on day 5. Repeat this procedure for all feeder cell batches tested.

On day 5, the culture medium was changed . CM2 with 3000 IU/mL IL-2 was prepared. 10 mL was needed for each culture flask. Using a 10 mL pipette, 10 mL of warm CM2 with 3000 IU/mL IL-2 was transferred to each culture flask. The culture flasks were returned to the incubator and incubated upright until day 7. The above procedure was repeated for all tested feeder cell batches.

Day 7, collect the culture . Remove the culture flasks from the incubator and transfer them to BSC, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove 10 mL of culture medium from each test flask and 15 mL of culture medium from each control flask.

Using a 10 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using the appropriate standard operating procedure with an automated cell counter. Record the counts on day 7. Repeat this procedure for all feeder cell batches tested.

The replication capacity of the feeder cell control flasks was insufficient, and the fold expansion of the culture flasks containing TILs was evaluated starting from day 0.

On day 7, continue processing the feeder cell control bottles until day 14. After counting the feeder cells on day 7, add 15 mL of fresh CM2 medium containing 3000 IU/mL IL-2 to each control bottle. Return the control bottles to the incubator and incubate them upright until day 14.

Day 14, extended non-proliferative phase of the feeder cell control flasks . Remove the culture flasks from the incubator and transfer them to BSCs, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove approximately 17 mL of culture medium from each control flask. Using a 5 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. Record the volume of each culture flask.

After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using an automated cell counter device following the appropriate standard operating procedure and record the count. Repeat this procedure for all feeder cell batches tested.

B. Results and Acceptance Criteria

Results . The dose of gamma irradiation was sufficient to prevent feeder cells from replicating. All batches were expected to meet the evaluation criteria and also showed a reduction in the total number of viable feeder cells remaining on day 7 of REP culture compared to day 0. All feeder cell batches were expected to meet the following evaluation criteria: 100-fold growth expansion of TILs up to day 7 of REP culture. The feeder cell control flask counts on day 14 were expected to continue the non-proliferative trend visible on day 7.

Acceptance Criteria . Each replicated TIL line for each batch of feeder cells must meet the following acceptance criteria. The acceptance criteria are doubled, as shown in Table 37 below.

Table 37. Implementation plan for acceptance criteria.

The study evaluated whether the irradiation dose was sufficient to prevent MNC feeder cells from replicating when cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. Insufficient replication capacity was assessed by total live cell count (TVC) as determined by automated cell counting at days 7 and 14 of the repetitive period (REP).

The acceptance criterion was "no growth," meaning that on days 7 and 14, the total number of viable cells did not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP.

The ability of feeder cells to support TIL expansion was evaluated. TIL growth was measured by the fold increase in live cells from day 0 of the Reproductive Phase (REP) to day 7 of the REP. On day 7, the TIL culture reached a minimum 100-fold expansion (i.e., more than 100 times the total number of live TIL cells introduced into the culture on day 0 of the REP), as evaluated by automated cell counting.

Emergency testing of MNC feeder cell batches that do not meet acceptance criteria . If an MNC feeder cell batch fails to meet any of the acceptance criteria outlined above, the following steps will be performed to retest the batch to rule out simple experimenter error as the cause.

If two or more satellite testing vials remain in the batch, then the batch shall be tested again. If one satellite testing vial remains in the batch, or if no satellite testing vials remain in the batch, then the batch is deemed unqualified according to the acceptance criteria listed above.

For the materials to be considered合格 (qualified), both the relevant batch and the control batch must meet the above acceptance criteria. Only after meeting these criteria is the batch permitted for use.

Example 4: Preparation of IL-2 stock solution

This embodiment describes a process for dissolving purified lyophilized recombinant human interleukin-2 in a stock sample suitable for use in other tissue culture protocols (including all those described in this application and in the embodiments), including those involving the use of rhIL-2.

Procedure. Prepare a 0.2% acetic acid solution (HAc). Transfer 29 mL of sterile water to a 50 mL conical tube. Add 1 mL of 1N acetic acid to the 50 mL conical tube. Mix thoroughly by inverting the tube 2-3 times. Sterilize the HAc solution by filtration using a Steriflip filter.

Prepare PBS containing 1% HSA. Add 4 mL of 25% HSA stock solution to 96 mL of PBS in a 150 mL sterile filter unit. Filter the solution. Store at 4°C. Complete the form for each vial of rhIL-2 prepared.

Prepare a stock solution of rhIL-2 (final concentration of 6 × ^10⁶ IU/mL). Each batch of rhIL-2 is different, and the required information can be found in the manufacturer's Certificate of Analysis (COA), such as: 1) the mass of rhIL-2 per vial (mg), 2) the specific activity of rhIL-2 (IU/mg), and 3) the recommended 0.2% HAc reduction volume (mL).

Calculate the required volume of 1% HSA for a batch of rhIL-2 using the following formula:

For example, according to the COA of rhIL-2 batch 10200121 (Cellgenix), the specific activity of a 1 mg vial is 25 × ^10⁶ IU/mg. It is recommended to reconstitute rhIL-2 in 2 mL of 0.2% HAc.

Wipe the rubber stopper of the IL-2 vial with an alcohol swab. Using a 16G needle attached to a 3mL syringe, inject the recommended volume of 0.2% HAc into the vial. Be careful not to remove the stopper while withdrawing the needle. Invert the vial three times and rotate until all powder dissolves. Carefully remove the stopper and place it on the alcohol swab. Add the calculated volume of 1% HSA to the vial.

Store rhIL-2 solution. For short-term storage (<72 hours), store vials at 4°C. For long-term storage (>72 hours), aliquot the vials into smaller volumes and store them in frozen vials at -20°C until ready for use. Avoid freeze/thaw cycles. Expiry date is 6 months after preparation date. The Rh-IL-2 label includes the supplier and catalog number, lot number, expiry date, operator initials, concentration, and aliquot volume.

Example 5: Cryopreservation Process

This embodiment describes a cryopreservation process for TILs prepared according to the procedures described herein, using a CryoMed controlled-rate cryostat (Thermo Scientific) of model 7454.

The equipment used is as follows: aluminum box holder (compatible with CS750 freezer bags), freezer box for 750mL bags, low-pressure (22psi) liquid nitrogen tank, freezer, thermocouple sensor (for the strip type of bag) and CryoStore CS750 freezer bags (OriGen Scientific).

From nucleation to -20°C, the freezing process provides a rate of 0.5°C and a cooling rate of 1°C/min until the final temperature of -80°C. The program parameters are as follows: Step 1—Wait at 4°C; Step 2: 1.0°C/min (sample temperature) to -4°C; Step 3: 20.0°C/min (chamber temperature) to -45°C; Step 4: 10.0°C/min (chamber temperature) to -10.0°C; Step 5: 0.5°C/min (chamber temperature) to -20°C; and Step 6: 1.0°C/min (sample temperature) to -80°C.

Example 6: Tumor amplification process using a defined culture medium

The procedures disclosed above can be performed using appropriate culture media (e.g., CTS ^™ OpTmizer ^™ T-cell expansion SFM, ThermoFisher, including, for example, DM1 and DM2) instead of CM1 and CM2 media.

Example 7: Exemplary GEN 2 preparation of cryopreserved TIL cell therapy

This embodiment describes cGMP manufacturing of Iovance Biotherapeutics' TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices. This embodiment describes an exemplary cGMP manufacturing of a TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices.

Table 38. Exemplary plans for process expansion.

Table 39. Culture Flask Volume

Day 0, CM1 medium preparation. In BSC, add reagents to RPMI 1640 culture bottles. Add the following reagents per bottle: heat-inactivated human AB serum (100.0 mL); GlutaMax ^™ (10.0 mL); gentamicin sulfate, 50 mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL).

Remove unnecessary materials from the BSC. Dispense culture medium reagents from the BSC, leaving gentamicin sulfate and HBSS in the BSC for use in preparing wash medium.

Thaw the IL-2 aliquot. Thaw a 1.1 mL IL-2 aliquot (6 × ^10⁶ IU/mL) (BR71424) until all ice has melted. Record the IL-2 batch number and expiration date.

Transfer the IL-2 stock solution to the culture medium. In BSC, transfer 1.0 mL of the IL-2 stock solution to the prepared CM1 Day 0 culture medium bottle. Add one bottle of CM1 Day 0 culture medium and 1.0 mL of IL-2 (6 × 10⁶ IU/mL).

Transfer G-REX100MCS to BSC. Aseptically transfer G-REX100MCS (W3013130) to BSC.

Pump all complete CM1 Day 0 culture medium into G-REX100MCS culture flasks. Alternatively, use conical tubes or G-Rex100MCS for tissue fragments.

Day 0, preparation of tumor washing medium. In BSC, 5.0 mL of gentamicin (W3009832 or W3012735) was added to a 1×500 mL HBSS medium (W3013128) bottle. Each bottle contained: HBSS (500.0 mL); gentamicin sulfate, 50 mg/mL (5.0 mL). Filtered HBSS containing gentamicin was prepared via a 1 L 0.22 μm filter unit (W1218810).

Day 0 Tumor Treatment. Obtain tumor samples and immediately transfer them to a kit at 2–8°C for treatment. Aliquot the tumor washing medium. Tumor washing 1 was performed using 8” forceps (W3009771). The tumor was removed from the sample vial and transferred to the prepared “Wash 1” culture dish. This was followed by tumor washing 2 and tumor washing 3. The tumor was measured and evaluated. It was assessed whether >30% of the total tumor area was observed to be necrotic and/or fatty tissue. Clean dissection was performed where applicable. If the tumor was large and >30% of the tissue surface was observed to be necrotic/fatty, “cleaning segmentation” was performed by removing the necrotic/fatty tissue using a combination of scalpel and/or forceps while preserving the internal structure of the tumor. The tumor was dissected. Using a combination of scalpel and/or forceps, the tumor sample was cut into an even number of appropriately sized fragments (up to 6 intermediate fragments). Intermediate tumor fragments were transferred. The tumor fragments were segmented into pieces approximately 3×3×3 mm in size. The intermediate fragments were stored to prevent dehydration. The intermediate fragment segmentation was repeated. The number of small pieces collected was determined. If only the desired tissue was preserved, additional favorable tumor fragments were selected from the “Favorable Intermediate Fragments” 6-well plate to fill the discarded fragments, so that up to 50 fragments were collected.

Prepare the conical tube. Transfer the tumor slice to a 50 mL conical tube. Prepare the BSC for G-REX100MCS. Remove the G-REX100MCS from the incubator. Aseptically transfer the G-REX100MCS culture flask to the BSC. Add the tumor fragment to the G-REX100MCS culture flask. Distribute the fragments evenly.

Incubate G-REX100MCS with the following parameters: G-REX culture flask: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Calculate: Incubation time; Lower limit = Incubation time + 252 hours; Upper limit = Incubation time + 276 hours.

After the process is complete, discard all remaining warm culture medium and thaw the aliquots of IL-2.

Day 11 - Culture medium preparation. Incubator monitoring. Incubator parameters: Temperature LED display: 37.0±2.0℃; CO2 percentage: 5.0±1.5% CO2.

Incubate 3 x 1000 mL RPMI 1640 medium (W3013112) bottles and 3 x 1000 mL AIM-V (W3009501) bottles in an incubator for ≥30 minutes. Remove the RPMI 1640 medium from the incubator. Prepare the RPMI 1640 medium. Filter the medium. Thaw 3 x 1.1 mL IL-2 aliquots (6 x 10⁶ IU/mL) (BR71424). Remove the AIM-V medium from the incubator. Add IL-2 to the AIM-V. Aseptically transfer the medium to the BSC using a 10 L Labtainer bag and relay pump transfer device.

Prepare a 10L Labtainer culture medium bag. Prepare a Baxa pump. Prepare another 10L Labtainer culture medium bag. Pump the culture medium into the 10L Labtainer. Remove the automatic pump tubing from the Labtainer bag.

Mix the culture medium. Gently knead the bag to mix. Sampling the culture medium according to the sampling plan. Take 20.0 mL of culture medium and place it in a 50 mL conical tube. Prepare the cell counting dilution tubes. In the BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" and lot number to four 15 mL conical tubes. Transfer the reagents from the BSC to 2–8°C. Prepare the 1 L transfer pack. On the outside of the BSC, fuse the 1 L transfer pack (according to process note 5.11) to the transfer device attached to the prepared "Complete CM2 Day 11 Medium" bag. Prepare the feeder cell transfer pack. Incubate the Complete CM2 Day 11 Medium.

Day 11 - TIL collection. Pretreatment form. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX100MCS from the incubator. Prepare 300mL transfer packs. Fuse the transfer packs to the G-REX100MCS.

Prepare the culture flask for TIL collection and begin the collection. Collect TILs. Using GatheRex, transfer the cell suspension through a blood filter to a 300 mL transfer pack. Examine the adherent cells on the membrane.

Rinse the culture flask membrane. Close the clamps on the G-REX100MCS. Ensure all clamps are closed. Heat-seal the TIL and "supernatant" transfer pack. Calculate the volume of the TIL suspension. Prepare the supernatant transfer pack for sampling.

Draw Bac-T samples. In the BSC, aspirate approximately 20.0 mL of supernatant from the 1 L “Supernatant” transfer pack and dispense into sterile 50 mL conical tubes.

Inoculate with BacT according to the sampling plan. Using an appropriately sized syringe, take 1.0 mL of sample from a prepared 50 mL conical tube labeled with BacT and inoculate it into an anaerobic bottle.

Incubate the TIL. Place the TIL transfer kit in an incubator only when needed. Perform cell counting and calculations. Measure the mean viable cell concentration and mean cell viability for each cell count. Viability ÷ 2. Viable cell concentration ÷ 2. Measure the upper and lower limits of the count. Lower limit: mean viable cell concentration × 0.9. Upper limit: mean viable cell concentration × 1.1. Confirm that both counts are within acceptable limits. Measure the mean viable cell concentration based on all four counts performed.

Adjusting the volume of the TIL suspension: Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume (A). Volume of the removed cell count sample (4.0 mL) (B) Adjusted total TIL cell volume C = A - B.

Calculate the total number of viable TIL cells. Average viable cell concentration*: total volume; total number of viable cells: C = A × B.

Flow cytometry calculation: If the total count of live TIL cells is ≥4.0× ^10⁷ , then calculate the volume of 1.0× ^10⁷ cells for the flow cytometry sample.

Total number of live cells required for flow cytometry: 1.0 × ^10⁷ cells. Cell volume required for flow cytometry: live cell concentration divided by 1.0 × ^10⁷ cells A.

Calculate the volume of a TIL suspension equal to 2.0 × ^10⁸ live cells. Calculate the volume of excess TIL cells to be removed as needed, and remove the excess TIL and place it in an incubator as needed. Calculate the total amount of excess TIL to be removed as needed.

The calculated volume of CS-10 medium added to the excess TIL cells at the target cell concentration for freezing is 1.0 × ^10⁸ cells/mL. Centrifuge the excess TIL as needed. Observe the conical tube and add CS-10.

Fill vials. Divide 1.0 mL of cell suspension into appropriately sized vials. Divide the remaining volume into appropriately sized vials. If the volume is ≤0.5 mL, add CS10 to the vials until the volume is 0.5 mL.

Calculate the cell volume required for 1 × ^10⁷ cells for cryopreservation. Remove the sample for cryopreservation. Place the TIL in an incubator.

Cryopreservation of samples. Observe the conical tube and slowly add CS-10, recording the volume of 0.5 mL of CS-10 added.

Day 11 - Feeder Cells. Obtain feeder cells. Obtain at least two bags of feeder cells from two different batches using an LN2 freezer. Preserve the cells on dry ice before thawing. Prepare a water bath or cryotherm. Thaw the feeder cells in a water bath or cryotherm at 37.0 ± 2.0 °C for approximately 3–5 minutes or until the ice has just melted. Remove the culture medium from the incubator. Combine the thawed feeder cells. Add the feeder cells to the transfer packs. Dispense the feeder cells from the syringe into the transfer packs. Mix the combined feeder cells and label the transfer packs.

Calculate the total volume of the feeder cell suspension in the transfer pack. Remove the cell counting samples. Using a separate 3 mL syringe, aspirate 4 × 1.0 mL of cell counting sample from the feeder cell suspension transfer pack using the non-essential injection port for each sample. Aliquot each sample into labeled cryovials. Perform cell counting, determine the multiplier, select a protocol, and enter the multiplier. Measure the mean viable cell concentration and mean cell viability for the cell count. Determine the upper and lower limits of count and confirm that they are within these limits.

Adjust the volume of the feeder cell suspension. Calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Calculate the total number of viable feeder cells. Obtain additional feeder cells as needed. Thaw additional feeder cells as needed. Place the fourth feeder cell bag in a zip-lock bag and thaw in a 37.0±2.0°C water bath or Cytotherm for approximately 3–5 minutes, and combine with the additional feeder cells. Measure the volume. Measure the volume of feeder cells in the syringe and record it below (B). Calculate the new total volume of feeder cells. Add the feeder cells to the transfer pack.

Prepare diluents as needed, adding 4.5 mL of AIM-V medium to four 15 mL conical tubes. Prepare for cell counting. Using a separate 3 mL syringe for each sample, remove 4 × 1.0 mL of the cell count sample from the feeder cell suspension transfer pack using the non-essential injection port. Perform cell counting and calculations. Determine the mean viable cell concentration based on all four counts performed. Adjust the feeder cell suspension volume and calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Subtract the removed 4.0 mL from the total feeder cell volume. Calculate the volume of feeder cell suspension required to obtain 5 x ^10⁹ viable feeder cells. Calculate the excess feeder cell volume. Calculate the volume of excess feeder cells to be removed. Remove the excess feeder cells.

Using a 1.0 mL syringe and a 16G needle, aspirate 0.15 mL of OKT3 and add OKT3. Heat-seal the feeder cell suspension transfer pack.

Day 11: G-REX Filling and Inoculation Setup for G-REX500MCS. Remove the "Complete CM2 Day 11 Medium" from the incubator and pump the medium into the G-REX500MCS. Pump 4.5L of medium into the G-REX500MCS, filling to the marked line on the culture flask. Heat seal as needed and incubate the culture flask. Fuse the feeder cell suspension transfer kit to the G-REX500MCS. Add feeder cells to the G-REX500MCS. Heat seal. Fuse the TIL suspension transfer kit to the culture flask. Add TIL to the G-REX500MCS. Heat seal. Incubate the G-REX500MCS at 37.0±2.0℃, CO2 percentage: 5.0±1.5% CO2.

Calculate the incubation window. Perform calculations to determine the appropriate time to remove the G-REX500MCS from the incubator on day 16. Lower limit: Incubation time + 108 hours. Upper limit: Incubation time + 132 hours.

Day 11: Cryopreservation of Excess TIL. Applicable to: Cryopreservation of excess TIL vials. Confirm that the Controlled Rate Freezer (CRF) has been set before freezing. Perform cryopreservation. Transfer the vial from the controlled rate freezer to the appropriate storage container. After freezing is complete, transfer the vial from the CRF to the appropriate storage container. Transfer the vial to the appropriate storage container. Record the storage location in LN2.

Day 16 Culture Medium Preparation. Preheat AIM-V medium. Calculate the warming time for culture bags 1, 2, and 3. Ensure all bags have been warmed for 12 to 24 hours. Set up the 10L Labtainer for supernatant. Attach the larger diameter end of the fluid pump transfer device to a concave port of the 10L Labtainer bag using a Luer connector. Set up and label the 10L Labtainer for supernatant. Set up the 10L Labtainer for supernatant. Ensure all clamps are closed before removing from the BSC. Note: Use the supernatant bag during TIL collection, which can be performed in parallel with culture medium preparation.

Thaw IL-2. Thaw 5 × 1.1 mL IL-2 aliquots (6 × ^10⁶ IU/mL) (BR71424) of each CTS AIM V medium bag until all ice melts. Aliquot 100.0 mL GlutaMax ^™ . Add IL-2 to GlutaMax ^™ . Prepare the CTS AIM V medium bag for preparation. Prepare the CTS AIM V medium bag for preparation. Multistage Baxa pump. Prepare the medium. Pump GlutaMax ^™ + IL-2 into the bag. Monitor parameters: Temperature LED display: 37.0 ± 2.0 °C, _CO₂ percentage: 5.0 ± 1.5% _CO₂ . Warm up the complete CM4 day 16 medium. Prepare the dilution.

Day 16, REP splitting. Incubator parameters monitored: Temperature LED display: 37.0±2.0℃, _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX500MCS from the incubator. Prepare a 1L transfer pack, label it TIL suspension, and weigh it as 1L.

The volume of the G-REX500MCS decreased. Approximately 4.5 L of culture supernatant was transferred from the G-REX500MCS to a 10 L Labtainer.

Prepare the culture flask for TIL collection. After removing the supernatant, close all clamps leading to the red tubing.

Initiate TIL collection . Vigorously tap the culture flask and rotate the culture medium to detach the cells, ensuring all cells are detached.

TIL Collection . Loosen all clamps leading to the TIL suspension transfer pack. Using GatheREX, transfer the cell suspension into the TIL suspension transfer pack. Note: Ensure the edges are kept tilted until all cells and culture medium are collected. Check for adherent cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS. Heat-seal the transfer pack containing TIL. Heat-seal the 10L Labtainer containing the supernatant. Record the weight of the transfer pack containing the cell suspension and calculate the suspension volume. Prepare the transfer pack for sample removal. Remove the test sample from the cell supernatant.

Aseptic and BacT test sampling . Take 1.0 mL of sample from the prepared 15 mL cone-labeled BacT. Take the cell counting sample. In the BSC, use a separate 3 mL syringe to take 4 × 1.0 mL cell counting samples from the TIL suspension transfer pack for each sample.

Remove the Mycoplasma sample. Using a 3 mL syringe, remove 1.0 mL from the TIL suspension transfer pack and place it into a prepared 15 mL conical tube labeled “Mycoplasma diluent”.

Prepare the transfer kit for inoculation. Place the TIL in an incubator. Remove the cell suspension from the BSC and place it in an incubator until needed. Perform cell counting and calculation. First, dilute the cell count sample to a 1:10 dilution by adding 0.5 mL of the cell suspension to 4.5 mL of prepared AIM-V medium. Measure the mean viable cell concentration and mean survival rate of the cells used for cell counting. Measure the upper and lower limits of counting. Note: Dilutions can be adjusted based on the expected cell concentration. Determine the mean viable cell concentration from all four counts performed. Adjust the volume of the TIL suspension. Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume minus the 5.0 mL removed for testing.

Calculate the total number of viable TIL cells. Calculate the total number of culture flasks to be inoculated. Note: The maximum number of G-REX500MCS culture flasks to be inoculated is five. If the calculated number of culture flasks to be inoculated exceeds five, inoculate only five culture flasks using all available volumes of cell suspension.

Calculate the number of culture flasks needed for subculture. Calculate the number of culture medium bags still needed besides the prepared bags. Prepare one 10L "CM4 Day 16 Medium" bag for every two G-REX-500M culture flasks as calculated. Continue inoculating the first G-REX-500M culture flask while preparing additional medium and warming it. Prepare the determined number of additional culture medium bags and warm them. Fill the G-REX500MCS. Prepare the pumpable medium and pump 4.5L of medium into the G-REX500MCS. Heat seal. Repeat filling. Incubate the culture flasks. Calculate the target volume of TIL suspension to be added to the new G-REX500MCS culture flasks. If the calculated number of culture flasks exceeds five, inoculate only five culture flasks using all volumes of cell suspension. Prepare the culture flasks for inoculation. Remove the G-REX500MCS from the incubator. Prepare the G-REX500MCS for pumping. Close all clamps except for the larger filter line. Remove the TIL from the incubator. Prepare the cell suspension for inoculation. Aseptically fuse the "TIL suspension" transfer package (according to process note 5.11) to the pump inlet line. Place the TIL suspension bag on the scale.

Inoculate the culture flasks with TIL suspension. Pump the calculated volume of TIL suspension into the culture flasks. Heat seal. Fill the remaining culture flasks.

Monitoring the incubator. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Incubation culture bottles.

The time range for removing G-REX500MCS from the incubator on day 22 was determined.

Day 22 Wash Buffer Preparation. Prepare a 10L Labtainer bag. Attach a 4” plasma transfer device to the 10L Labtainer bag via a Luer connector in the BSC. Prepare the 10L Labtainer bag. Close all clamps before transferring out of the BSC. Note: Prepare one 10L Labtainer bag for every two G-REX 500MCS culture flasks to be collected. Aspirate the Plasmalyte pump into the 3000mL bag and remove air from the 3000mL Origen bag by inverting the pump and manipulating the bag's position. Add 25% human albumin to the 3000mL bag. Obtain a final volume of 120.0mL of 25% human albumin.

Prepare the IL-2 diluent. Using a 10 mL syringe, remove 5.0 mL of LOVO wash buffer through the needle-free injection port on the LOVO wash buffer bag. Dispense the LOVO wash buffer into 50 mL conical tubes.

Distribute LOVO wash buffer into equal portions of the CRF blank bags. Using a 100mL syringe, draw 70.0mL of LOVO wash buffer from the needleless injection port.

Thaw one 1.1 mL IL-2 (6 × 10⁶ IU/mL) until all ice has melted. Add 50 μL of IL-2 stock solution (6 × ^10⁶ IU/mL) to a 50 mL conical tube labeled “IL-2 Diluent”.

Preparation for cryopreservation . Place 5 cryopreservation boxes at 2-8°C to pretreat them for use in the final product cryopreservation.

Prepare the cell counting dilution. In a BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" to four separate 15 mL conical tubes. Prepare for cell counting. Label the four frozen vials with vial numbers (1-4). Store the vials in the BSC for later use.

TIL collection on day 22. Monitor the incubator. Incubator parameters: Temperature LED display: 37±2.0℃, CO2 percentage: 5%±1.5%. Remove the G-REX500MCS culture flask from the incubator. Prepare and label the TIL collection bag. Seal any additional connectors. Volume reduction: Transfer approximately 4.5L of supernatant from the G-REX500MCS to the supernatant bag.

Prepare the culture flask for TIL collection. Initiate TIL collection. Vigorously tap the culture flask and rotate the culture medium to detach the cells. Ensure all cells have detached. Initiate TIL collection. Loosen all clamps leading to the TIL suspension collection bag. TIL collection. Using GatheRex, transfer the TIL suspension to a 3000 mL collection bag. Examine the adhered cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS, ensuring all clamps are closed. Transfer the cell suspension to the LOVO source bag. Close all clamps. Heat seal. Remove 4 × 1.0 mL cell counting samples.

Perform cell counting. Cell counting and calculation were performed using NC-200 and process note 5.14. The cell counting sample was first diluted by adding 0.5 mL of cell suspension to 4.5 mL of prepared AIM-V medium, resulting in a 1:10 dilution. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The upper and lower limits of counting were determined. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The LOVO source bag was weighed. The total number of viable TIL cells was calculated. The total number of nucleated cells was calculated.

Prepare the Mycoplasma diluent. Take 10.0 mL from a supernatant bag via the Luhr sample port and place it into a 15 mL conical tube.

Perform the "TIL G-REX collection" protocol and determine the target volume of the final product. Load the disposable kit. Remove the filtrate bag. Enter the filtrate volume. Place the filtrate container on the lab bench. Attach PlasmaLyte. Confirm that PlasmaLyte has been attached and that it is moving. Attach the source container to the tubing and confirm that it has been attached. Confirm that PlasmaLyte is moving.

Final preparation and filling. Target volume/bag calculation. Calculate the volume of CS-10 and LOVO wash buffer to be prepared in blank bags. Prepare CRF blank bags.

Calculate the volume of IL-2 to be added to the final product. Required final IL-2 concentration (IU/mL) - 300 IU/mL. IL-2 working stock solution: 6 × ^10⁴ IU/mL. Assemble and connect the device. Aseptically fuse the 4S-4M60 to the CC2 unit connector. Aseptically fuse the CS750 cryoprotectant bag to the prepared wire bundle. Fuse the CS-10 bag to the tip of the 4S-4M60. Prepare TILs with IL-2. Using an appropriately sized syringe, extract the measured amount of IL-2 from the “IL-2 6 × ^10⁴ ” aliquot. Label the prepared TIL bag. Add the prepared TIL bag to the device. Add CS10. Switch syringes. Aspirate approximately 10 mL of air into a 100 mL syringe and replace the 60 mL syringe on the device. Add CS10. Prepare the CS-750 bag. Dispense cells.

Remove air from the final product bag and obtain the retention. Once the last final product bag has been filled, close all clamps. Draw 10 mL of air into a new 100 mL syringe and replace the syringe on the device. Dispense the retention into 50 mL conical tubes and label the tubes “Retention” and the batch number. Repeat the air removal steps for each bag.

Prepare the final product for cryopreservation, including visual inspection. Store the cryopreservation bag on a cooling pack or at 2-8°C before cryopreservation.

Remove the cell counting sample. Using an appropriately sized pipette, remove 2.0 mL of the retained sample and place it in a 15 mL conical tube for cell counting. Perform cell counting and calculation. Note: Only dilute one sample to an appropriate confirmatory dilution. Dilute the remaining sample to the appropriate dilution factor and continue counting. Measure the mean viable cell concentration and mean viability of the cells used for cell counting. Measure the upper and lower limits of count. Note: The dilution may be adjusted according to the expected cell concentration. Measure the mean viable cell concentration and mean viability. Measure the upper and lower limits of count. Calculate IFN-γ. Heat-seal the final product bag.

The following exemplary sampling plan is used to label and collect samples.

Table 40. Sample Plan.

Sterility and BacT test . Test sampling. In the BSC, take 1.0 mL of sample from the retained cell suspension collected using an appropriately sized syringe and inoculate it into an anaerobic flask. Repeat the above steps for the aerobic flask.

Cryopreservation of the final product. Prepare the controlled-rate freezer (CRF). Verify that the CRF is set up. Set the CRF probe. Place the final product and sample in the CRF. Measure the time required to reach 4℃ ± 1.5℃ and continue the CRF operation. Complete the CRF and store. Stop the CRF after completion. Remove the box and vial from the CRF. Transfer the box and vial to gas chromatography-tandem nitrogen (LN2) for storage. Record the storage location.

The final processing and analysis of the drug included the following tests: (Day 22) CD3+ cell count of REP on Day 22 by flow cytometry; (Day 22) Gram staining method (GMP); (Day 22) bacterial endotoxin test by gel clot LAL assay (GMP); (Day 16) BacT sterility assay (GMP); (Day 16) TD-PCR (GMP) detection of Mycoplasma DNA; acceptable appearance characteristics; (Day 22) BacT sterility assay (GMP); (Day 22) IFN-γ assay. Other potency assays as described herein were also used to analyze the TIL product.

Example 8: Exemplary Implementation of the GEN 3 Amplification Platform

Day 0

Prepare tumor washing medium. Warm the medium before starting. Add 5 mL of gentamicin (50 mg/mL) to a 500 mL HBSS bottle. Add 5 mL of tumor washing medium to a 15 mL Erlenmeyer flask for OKT3 dilution. Prepare feeder cell bags. Aseptically transfer feeder cells to feeder cell bags and store at 37°C until use or freeze. If stored at 37°C, count the feeder cells. If frozen, thaw and then count the feeder cells.

The optimal range for feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with a 4.5 mL LAIM-V. Add 0.5 mL of cell fraction for each cell count. If the total number of viable feeder cells is ≥ 1 × ^10⁹ cells, adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag to add 1 × ^10⁹ cells to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of tumor washing medium to an OKT3 aliquot (100 μL). Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the second feeder cell bag. Adjust the medium volume to a total volume of 2 L. Transfer the second feeder cell bag to an incubator.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 0: 15 μg/vial, i.e., 30 ng/mL in 500 mL, up to approximately 60 μL, 1 aliquot.

Add 5 mL of tumor wash medium to all wells of a 6-well plate labeled as excess tumor flakes. Reserve the tumor wash medium for further use in maintaining tumor hydration during dissection. Add 50 mL of tumor wash medium to each 100 mm Petri dish.

Using a ruler as a reference, divide the tumor into ^27mm segments (3×3×3mm) under the dissecting culture dish lid. Dissect the intermediate segments until 60 segments are obtained. Count the total number of final segments and prepare G-REX-100MCS culture flasks (usually 60 segments/flask) based on the number of final segments produced.

Preserve favorable tissue fragments in conical tubes labeled Fragment Tubes 1 through 4. Calculate the number of G-REX-100MCS culture flasks seeded with feeder cell suspension based on the number of initial fragment tubes.

Remove the feeder cell bag from the incubator and inoculate with G-REX-100 MCS. Label it D0 (day 0).

Add the tumor fragment to the culture in the G-REX-100 MCS . Under aseptic conditions, unscrew the caps of the G-REX-100 MCS labeled "Tumor Fragment Culture (D0) 1" and the 50 mL conical tube labeled "Fragment Tube". Rotate the opened fragment tube 1 while simultaneously slightly lifting the cap of the G-REX-100 MCS. Add the culture medium and fragment together to the G-REX-100 MCS while rotating. Record the number of fragments transferred to the G-REX-100 MCS.

Once the fragment is at the bottom of the GREX culture flask, aspirate 7 mL of culture medium and create seven 1 mL aliquots: 5 mL for extended characterization and 2 mL for sterility. Store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until needed.

Inoculate one anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle with 1 mL of the final fragment culture supernatant. Repeat the above procedure for each culture bottle from which samples are taken.

On the 7th-8th day

Prepare the feeder cell bags. If frozen, thaw the feeder cell bags in a 37°C water bath for 3–5 minutes. If frozen, count the feeder cells. The optimal feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with 4.5 mL AIM-V. For each cell count, add 0.5 mL of the cell fraction to a new frozen tube. Mix the sample thoroughly and perform cell counting.

If the total number of live feeder cells is ≥2× ^10⁹ cells, proceed to the next step to adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag so that 2× ^10⁹ cells can be added to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of HBSS to 100 μL of OKT3 aliquot. Mix by pipetting three times, upwards and downwards. Prepare two aliquots.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 7/8: 30 μg/vial, i.e., 60 ng/mL in 500 mL, up to 120 μL, 2 aliquots.

Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the feeder cell bag, ensuring all is added. Adjust the culture medium volume to a total of 2 L. Repeat the above steps for the second OKT3 aliquot and add it to the feeder cell bag. Transfer the second feeder cell bag to an incubator.

Prepare G-REX100MCS culture flasks containing feeder cell suspension . Record the number of G-REX100MCS culture flasks to be processed based on the number of G-REX culture flasks produced on day 0. Remove the G-REX culture flasks from the incubator and remove the second feeder cell bag from the incubator.

Remove the supernatant before adding the feeder cell suspension . Connect a 10 mL syringe to a G-REX100 culture flask and aspirate 5 mL of culture medium. Prepare five 1 mL aliquots, 5 mL for extended characterization, and store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until requested by the initiator. Label each G-REX100 culture flask and repeat the above steps.

Depending on the number of culture flasks, 5-20 × 1 mL samples are used for characterization:

●5mL = 1 culture flask

●10mL = 2 culture flasks

●15mL = 3 culture flasks

●20mL = 4 culture flasks

Continue seeding feeder cells into G-REX100 MCS flasks, and repeat the above procedure for each G-REX100 MCS flask. Using aseptic transfer, transfer 500 mL of the second feeder cell bag by weight (assuming 1 g = 1 mL) to each G-REX-100 MCS flask and record the transfer volume. Label this as Day 7 culture and repeat the above procedure for each G-REX100 flask. Transfer the G-REX-100 MCS flasks to an incubator.

Days 10-11

Remove the first G-REX-100MCS culture flask and, under aseptic conditions, use a 10mL syringe to extract 7mL of pretreated culture supernatant. Prepare seven 1mL aliquots: 5mL for extended characterization and 2mL for sterile samples.

Carefully mix the culture flasks and use a new 10mL syringe to remove 10mL of the supernatant and transfer it to a 15mL tube labeled D10/11 Mycoplasma supernatant.

Carefully mix the culture flasks and, using a new syringe, dispense the following volumes based on the number of culture flasks to be processed:

● 1 bottle = 40mL

●2 bottles = 20mL/bottle

● 3 bottles = 13.3 mL/bottle

● 4 bottles = 10mL/bottle

Aspirate a total of 40 mL from all culture flasks and collect the samples in a 50 mL conical tube labeled ‘Day 10/11 QC Sample’. Store the tube in an incubator until needed. Perform cell counting and cell distribution.

Five aliquots (pretreated culture supernatant) used for extended characterization were stored at ≤-20°C until needed. One anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle were inoculated with 1 mL of pretreated culture supernatant, respectively.

Continue transferring the cell suspension to G-REX-500MCS and repeat the above procedure for each G-REX-100MCS. Under aseptic conditions, transfer the contents of each G-REX-100MCS to a G-REX-500MCS, monitoring approximately 100 mL of fluid transfer each time. Stop transferring when the volume of the G-REX-100MCS decreases to 500 mL.

During the transfer step, use a 10 mL syringe and aspirate 10 mL of cell suspension from the G-REX-100MCS into the syringe. Follow the instructions according to the number of flasks in culture. If only 1 flask: use two syringes to aspirate a total of 20 mL. If 2 flasks: aspirate 10 mL from each flask. If 3 flasks: aspirate 7 mL from each flask. If 4 flasks: aspirate 5 mL from each flask. Transfer the cell suspension to a common 50 mL conical tube. Keep in the incubator until the cell counting step and QC sample. The total number of cells required for QC is approximately 20e6 cells: 4 × 0.5 mL cell counts (cell counts are undiluted first).

The required cell quantity for the assay is as follows:

1. At least 10 × ^10⁶ cells should be used for efficacy assays, such as those described herein, or for IFN-γ or granzyme B assays.

2.1 × ^10⁶ cells were used for Mycoplasma.

3.5 × ^10⁶ cells were used for CD3+/CD45+ flow cytometry.

Transfer the G-REX-500MCS culture flask to an incubator.

Prepare QC samples . In this embodiment, at least 15 × ^10⁸ cells are required for the assay. Assays include: cell count and viability; Mycoplasma (1 × ^10⁶ cells/mean viable concentration); flow cytometry (5 × ^10⁶ cells/mean viable concentration); and IFN-γ assay (5 × ^10⁶ cells - 1 × ^10⁶ cells; IFN-γ assay requires 8-10 × ^10⁶ cells).

Calculate the volume of the cell fraction cryopreserved at 10 × ^10⁶ cells/mL, and calculate the number of vials required.

Days 16-17

Wash buffer preparation (1% HSA Plasmalyte A). Transfer the HSA and Plasmalyte to a 5L bag to prepare the LOVO wash buffer. Under aseptic conditions, transfer a total volume of 125 mL of 25% HSA to a 5L bag. Remove 10 mL or 40 mL of the wash buffer and transfer it to an 'IL-2 6× ^10⁴ IU/mL tube' (10 mL if the IL-2 is pre-prepared, or 40 mL if the IL-2 is freshly prepared).

Calculate the volume of restored IL-2 added to Plasmalyte + 1% HSA: Volume of restored IL-2 = (Final concentration of IL-2 × Final volume) / Specific activity of IL-2 (based on standard determination). The final concentration of IL-2 is 6 × ^10⁴ IU/mL. The final volume is 40 mL.

Take the calculated initial volume of IL-2 required for IL-2 recovery and transfer it to a tube labeled 'IL-2 6 × ^10⁴ IU/mL'. Add 100 μL of IL-2 6 × ^10⁶ IU/mL from the pre-prepared aliquot to a tube labeled 'IL-2 6 × ^10⁴ IU/mL' containing 10 mL of LOVO wash buffer.

Remove approximately 4500 mL of supernatant from the G-REX-500MCS culture flask. Rotate the remaining supernatant and transfer the cells to the cell collection pocket. Repeat the above steps for all G-REX-500MCS culture flasks.

Take 60 mL of the supernatant and add it to the supernatant tube for quality control determination, including mycoplasma detection. Store at +2–8°C.

Cell collection . Cell counting. Prepare four 15 mL conical tubes containing 4.5 mL of AIM-V. These conical tubes can be prepared in advance. Optimal range = between 5 × ^10⁴ and 5 × ^10⁶ cells/mL (1:10 dilution recommended). For a 1:10 dilution, add 500 μL of CF to the previously prepared 4500 μL of AIM-V. Record the dilution factor.

Calculate the total cells (TC) of pre-LOVO (survival + death) =

Average total cells

Concentration (pre-LOVO TC concentration)

(Survival + Death)

X

Volume of source bag

Calculate the TVC (total viable cells) of pre-LOVO (survival) =

Average total viable cells

Concentration (pre-LOVO TVC)

(Survival)

X

LOVO source bag volume

When the total cell count (TC) is >5 × ^10⁹ , remove 5 × ^10⁸ cells for cryopreservation as a reserve sample for MDA. 5 × ^10⁸ ÷ average TC concentration (step 14.44) = volume to be removed.

When the total cell count (TC) is ≤5× ^10⁹ , 4× ^10⁶ cells are removed and cryopreserved as a reserve sample for MDA. 4× ^10⁶ ÷ average TC concentration = volume to be removed.

When determining the total cell count, the number of cells to be removed should allow for the retention of 150 × ^10⁹ viable cells. Confirm the pre-LOVO TVC as 5 × ^10⁸ or 4 × ^10⁶ , or not applicable. Calculate the volume of cells to be removed.

Calculate the remaining total cells in the bag. Calculate the pre-LOVO TC (total cells). [Average total cell concentration x remaining volume = pre-LOVO remaining TC]

Select the corresponding process from Table 41 based on the total number of remaining cells.

Table 41. Total number of cells.

Select the volume of IL-2 to be added corresponding to the procedure used. The volume is calculated as follows: Residue volume × 2 × 300 IU/mL = Required IU of IL-2. Required IU of IL-2 / 6 × ^10⁴ IU/mL = Volume of IL-2 added after LOVO bagging. Record all added volumes. Obtain samples in frozen vials for further analysis.

Mix the cell products thoroughly. Seal all bags for further processing, including freezing where appropriate.

Perform endotoxin, IFN-γ, sterility, and other assays on the obtained frozen vial samples as needed.

Example 9: Exemplary Processes for GEN 2 and GEN 3

This example illustrates the Gen 2 and Gen 3 processes. The Gen 2 and Gen 3 TILs typically consist of autologous TILs derived from individual patients (via surgical tumor resection) and then expanded ex vivo. The first expansion step in initiating the Gen 3 process involves cell culture in the presence of interleukin-2 (IL-2) and the monoclonal antibody OKT3, which targets the T-cell co-receptor CD3 on the cytoskeleton of irradiated peripheral blood mononuclear cells (PBMCs).

The fabrication of the Gen 2 TIL product consists of two stages: 1) pre-rapid expansion (pre-REP) and 2) rapid expansion protocol (REP). During pre-REP, excised tumors are cut into ≤50 fragments of 2-3 mm in each dimension. These fragments are cultured for 11 days in serum-containing medium (RPMI 1640 medium supplemented with 10% HuSAB) and 6,000 IU/mL interleukin-2 (IL-2). On day 11, TILs are collected and introduced into a large-scale secondary REP expansion. The REP consists of the following: activation of ≤200 × ^10⁶ live cells from pre-REP for 5 days in ⁵ L of CM2 supplemented with 3,000 IU/mL rhIL-2, with the feeder cells loaded with 150 μg of monoclonal anti-CD3 antibody (OKT3). On day 16, the culture volume was reduced by 90%, and the cells were fractionated to ≥1× ^10⁹ viable lymphocytes/flask into multiple G-REX-500 culture flasks, and brought to a final volume of 5L with CM4. The TIL was then incubated for another 6 days. On day 22, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

The fabrication of Gen 3 TIL products consists of three stages: 1) initiation of the first expansion protocol, 2) rapid second expansion protocol (also known as the rapid expansion phase or REP), and 3) dissociation of subcultures. To achieve initiation of the first expansion TIL proliferation, the excised tumor was fragmented into ≤120 fragments, each 2-3 mm in dimensions. On day 0 of initiation of the first expansion, a feeder layer of approximately 2.5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was established on a surface area of approximately 100 ^cm² in each of three 100 MCS dishes. The tumor fragments were distributed in the three 100 MCS dishes and cultured for 7 days in CM1 medium containing 500 mL of serum and 6,000 IU/mL of interleukin-2 (IL-2) and 15 μg of OKT-3. On day 7, REP was initiated as follows: an additional feeder cell layer of approximately 5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was incorporated into the tumor fragmentation culture phase in each of three 100 MCS dishes and cultured with 500 mL CM2 medium and 6,000 IU/mL IL-2 and 30 μg OKT-3. REP initiation was enhanced by activating the entire initiation first expansion culture in the same dish, which was achieved by transferring OKT3-loaded feeder cells to the 100 MCS dishes using a closed-system fluid transfer. For Gen 3, TIL scaling up or splitting involved the following procedural steps: the entire cell culture was scaled up and transferred to the larger dish via a closed-system fluid transfer (from a 100 M culture flask to a 500 M culture flask) and an additional 4 L CM4 medium was added. On day 16, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

Overall, the Gen 3 process is a shorter, more scalable, and easier-to-modify amplification platform that will be suitable for stable manufacturing and process comparability.

Table 50. Comparison of the manufacturing processes of Exemplary Gen 2 and Exemplary Gen 3.

On day 0, for both processes, tumor cells were washed three times and fragments were randomly grouped into two pools; one pool for each process. For the Gen 2 process, fragments were transferred to a GREX 100MCS flask containing 1 LCM1 of medium with 6,000 IU/mL rhIL-2. For the Gen 3 process, fragments were transferred to a G-REX100MCS culture flask containing 500 mL CM1 of medium with 6,000 IU/mL rhIL-2, 15 μg OKT-3, and 2.5 × ^10⁸ feeder cells. TILs were inoculated on Rep start day according to different days for each process. For the Gen 2 process, where the volume of the G-REX-100MCS culture flasks decreased by 90%, the collected cell suspension was transferred to new G-REX-500MCS on day 11 to initiate REP in CM2 medium containing IL-2 (3000 IU/mL) plus 5 × ^10⁹ feeder cells and OKT-3 (30 ng/mL). Cells were expanded according to the protocol and split into multiple G-REX-500MCS culture flasks containing CM4 medium and IL-2 (3000 IU/mL) on day 16. Cultures were then collected and cryopreserved on day 22 according to the protocol. For the Gen 3 process, REP initiation was performed on day 7 using the same G-REX-100MCS. In short, 500 mL of CM2 medium containing IL-2 (6000 IU/mL), 5 × ^10⁸ feeder cells, and 30 μg OKT-3 was added to each culture flask. On days 9–11, scale up the culture. Transfer the entire volume of G-REX100M (1 L) to G-REX-500MCS and add 4 L of CM4 containing IL-2 (3000 IU/mL). Incubate the flask for 5 days. On day 16, collect the culture and freeze it.

The comparison included three different tumors: two lung tumors (L4054 and L4055) and one melanoma tumor (M1085T).

For L4054 and L4055, CM1 (medium 1), CM2 (medium 2), and CM4 (medium 4) media were prepared in advance and maintained at 4°C. CM1 and CM2 media were prepared without filtration to compare cell growth with and without filtration of the media.

For L4055 tumors, the culture medium was heated at 37°C for up to 24 hours during REP initiation and scaling up.

Results . For the total number of viable cells achieved, the results for Gen 3 were within 30% of those for Gen 2. Upon restimulation, the Gen 3 final product exhibited higher IFN-γ production. As measured by the total number of unique CDR3 sequences present, the Gen 3 final product showed increased clonal diversity. The Gen 3 final product also exhibited a longer mean telomere length.

Pre-REP and REP expansion in Gen 2 and Gen 3 processes followed the procedures described above. For each tumor, both pools contained an equal number of fragments. Due to the smaller size of the tumors, the maximum number of fragments per flask could not be reached. Total pre-REP cells (TVCs) were collected and counted on day 11 of the Gen 2 process and day 7 of the Gen 3 process. To compare the two pre-REP groups, the cell count was divided by the number of fragments provided in the culture to calculate the average number of viable cells per fragment. As indicated in Table 51 below, more cells were consistently grown per fragment in the Gen 2 process compared to the Gen 3 process. The extrapolation of the expected TVC number on day 11 of the Gen 3 process was calculated by dividing the pre-REP TVC by 7 and then multiplying by 11.

Table 51. Pre-REP cell count

*L4055, unfiltered culture medium.

For both Gen 2 and Gen 3 processes, TVCs were counted according to process conditions, and the percentage of viable cells per day of the process was generated. At harvest, cells were collected on day 22 (Gen 2) and day 16 (Gen 3), and TVC counts were generated. The TVCs were then divided by the number of fragments provided on day 0 to calculate the average number of viable cells per fragment. The fold increase was calculated by dividing the collected TVCs by the REP initiation TVCs. As shown in Table 52, comparing Gen 2 and Gen 3, the fold increases were similar for L4054; in the case of L4055, the fold increase was higher for the Gen 2 process. Specifically, in this case, the medium was heated to 24°C prior to the REP initiation day. A higher fold increase was also observed in Gen 3 for M1085T. The extrapolation of the expected TVC count on day 22 of the Gen 3 process was calculated by dividing the REP TVCs by 16 and then multiplying by 22.

Table 52. Total viable cell count and fold expansion of TIL final products.

*L4055, unfiltered culture medium.

Table 53: Survival percentage of final TIL products: Final TIL REP products were compared to the survival percentage release criteria after collection. All conditions for Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Following collection, the final TIL REP product was compared against release criteria based on survival percentage. All conditions for both Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Table 53. Survival Percentage of REP (TIL Final Product)

Because the number of fragments in each vial is less than the maximum required number, the estimated cell count on the collection day is calculated for each tumor. This assessment is based on the expectation that the clinical tumor is large enough on day 0 to be inoculated with 2 or 3 vials.

Table 54. Extrapolation of estimated cell counts to full-size 2 and 3 bottles for the Gen 3 process.

Immunophenotypic Analysis – Comparison of Phenotypic Markers in TIL End Products. Three tumor types, L4054, L4055, and M1085T, underwent TIL amplification during both Gen 2 and Gen 3 processes. Following collection, REP TIL end products were analyzed by flow cytometry to test for purity, differentiation, and memory markers. For all conditions, the percentage of TCR a/b+ cells exceeded 90%.

TILs collected from the Gen 3 process showed higher expression of CD8 and CD28 compared to those collected from the Gen 2 process. The Gen 2 process showed a higher percentage of CD4+.

TILs collected from the Gen 3 process showed higher expression of the central memory chamber compared to TILs collected from the Gen 2 process.

Activation and depletion biomarkers were analyzed in TILs from two tumors, L4054 and L4055, to compare the final TIL products from the Gen 2 and Gen 3 TIL amplification processes. Activation and depletion biomarkers for the Gen 2 and Gen 3 processes were similar.

Interferon-γ secretion upon restimulation. On the collection days, day 22 of Gen 2 and day 16 of Gen 3, TILs of L4054 and L4055 were restimulated overnight using coated anti-CD3 plates. M1085T was restimulated using anti-CD3, CD28, and CD137 beads. Under all conditions, the supernatant was collected and frozen 24 hours after restimulation. IFNγ analysis by ELISA was performed simultaneously on the supernatants from both processes using the same ELISA plate. Higher IFNγ production from the Gen 3 process was observed in all three tumors analyzed.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption during Gen 2 versus Gen 3 processes, cell supernatants were collected on REP initiation, scale-up, and collection days for tumors L4054 and L4055. The amount of IL-2 in the cell culture supernatant was measured using a Quantitate ELISA kit from R&D. A general trend indicates that IL-2 concentrations remained higher during Gen 3 compared to Gen 2. This may be attributed to the higher IL-2 concentration at Gen 3 REP initiation (6000 IU/mL) and residual culture medium throughout the process.

Metabolic substrate and metabolite analysis. The levels of metabolic substrates (e.g., D-glucose and L-glutamine) are measured as substitutes for overall culture medium consumption. Their reciprocal metabolites, such as lactate and ammonia, are also measured. Glucose is a monosaccharide in the culture medium, used by mitochondria to produce energy in the form of ATP. When glucose is oxidized, lactate is produced (lactate is an ester of lactate). Lactate is produced in large quantities during the exponential growth phase of cells. High levels of lactate can negatively impact cell culture processes.

For Gen 2 and Gen 3 processes, used culture media from L4054 and L4055 were collected on REP start, scale-up, and collection days. Used culture media were collected on days 11, 16, and 22 of Gen 2, and on days 7, 11, and 16 of Gen 3. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

L-Glutamine is an unstable essential amino acid required in cell culture media formulations. Glutamine contains an amine, and this amide group allows for the transport and delivery of nitrogen into cells. When L-glutamine is oxidized, cells produce toxic ammonia byproducts. To counteract L-glutamine degradation, GlutaMax ^™ was supplemented into the culture medium during the Gen 2 and Gen 3 processes, as it is more stable in aqueous solution and does not spontaneously degrade. In both tumor lines, the Gen 3 group showed a decrease in L-glutamine and GlutaMax ^™ during the process, as well as an increase in ammonia throughout the REP. In the Gen 2 group, constant L-glutamine and GlutaMax ^™ concentrations were observed, along with a slight increase in ammonia production. For ammonia, the Gen 2 and Gen 3 processes were similar at the collection day, showing only slight differences in L-glutamine degradation.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats at L4054 and L4055 was measured using Flow-FISH technology during Gen 2 and Gen 3. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Gen 3 showed similar telomere lengths to Gen 2.

CD3 analysis. To determine the clonal diversity of cell products generated in each process, the collected TIL final products from L4054 and L4055 were sampled and analyzed by sequencing the CDR3 portion of the T cell receptor for clonal diversity analysis.

Table 55 shows a comparison of the percentage of unique CDR3 sequences shared on L4054 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 199 sequences with Gen 2 final products, and 97.07% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 55. Comparison of the common uCDR3 sequence between Gen 2 and Gen 3 processes on L4054.

Table 56 shows a comparison of the percentage of unique CDR3 sequences shared on L4055 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 1833 sequences with Gen 2 final products, and 99.45% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 56. Comparison of common uCDR3 sequences between Gen 2 and Gen 3 processes on L4055.

CM1 and CM2 culture media were prepared in advance without filtration and kept at 4°C until used for tumor L4055 for Gen 2 and Gen 3 processes.

For L4055 tumors, on the REP initiation day, the culture medium was heated at 37°C for 24 hours for use in the Gen 2 and Gen 3 processes.

LDH was not measured in the supernatant collected during the process.

M1085T TIL cells were counted using a K2 cellometer.

On the tumor M1085T, samples are unavailable, such as supernatant for metabolic analysis; TIL products for activation and depletion marker analysis, telomere length and CD3-TCR vb analysis.

Conclusion . This example compares three independent donor tumor tissues based on functional quality attributes, expanded phenotypic characterization of the Gen 2 and Gen 3 processes, and culture medium consumption.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

Assuming pre-REP collection is performed on day 11 instead of day 7 and REP collection is performed on day 22 instead of day 16, extrapolated cell numbers for the Gen 3 process were calculated. In both cases, Gen 3 showed closer TVC numbers compared to the Gen 2 process, indicating that early activation enhances TIL growth.

Extrapolating values from other flasks (2 or 3) in the Gen 3 process, assuming larger tumor sizes and the maximum number of fragments required for each process as described. It was observed that collections performed on day 16 of the Gen 3 process achieved similar TVC doses compared to day 22 of the Gen 2 process. This observation is significant and indicates that early activation of the culture may reduce TIL treatment time.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

In terms of phenotypic characterization, higher CD8+ and CD28+ expression was observed in all three tumors during the Gen 3 process compared to the Gen 2 process.

Compared to the Gen 2 process, the Gen 3 process shows a slightly higher central memory compartment.

Although the Gen 3 process is shorter in duration, the Gen 2 and Gen 3 processes show similar activation and exhaustion markers.

In the three tumors analyzed, the IFNγ production on the final product of Gen 3 was 3-fold higher than that of Gen 2. This data suggests that the Gen 3 process produces a more potent and efficient TIL product compared to the Gen 2 process, which may be attributed to higher expression of CD8 and CD28 on Gen 3. Phenotypic characterization showed a positive trend in CD8+ and CD28+ expression in Gen 3 compared to the Gen 2 process in all three tumors.

The telomere lengths of the final TIL products from Gen 2 and Gen 3 are similar.

The glucose and lactate levels of the final products of Gen 2 and Gen 3 were similar, indicating that the nutrient levels on the culture medium were not affected during the Gen 3 process because no reduction removal was performed each day during the process and the overall culture medium volume was smaller compared to Gen 2.

Compared to the Gen 2 process, the processing time of the entire Gen 3 process is reduced by about half, which will significantly reduce the cost of goods sold (COG) of TIL products amplified through the Gen 3 process.

IL-2 consumption indicates the general trend of IL-2 consumption during the Gen 2 process, and that IL-2 is higher during the Gen 3 process due to the lack of removal of old culture medium.

CDR3 TCRab sequence analysis revealed high clonal diversity in the Gen 3 process.

Adding feeder cells and OKT-3 on day 0 of the pre-REP allows for early activation of TILs and enables TIL growth using the Gen 3 process.

Table 57 describes the various implementation schemes and results of the Gen 3 process compared to the current Gen 2 process.

Table 57. Exemplary Gen 3 process characteristics.

Example 10: Exemplary GEN 3 process (also known as GEN 3.1)

This embodiment describes further studies regarding the comparability between Gen 2 and Gen 3 processes for TIL amplification. The Gen 3 process is modified to include an activation step early in the process, thereby increasing the final total live cells (TVC) output while maintaining phenotypic and functional profile. As described below, the Gen 3 implementation is modified to another implementation, referred to herein as Gen 3.1.

In some implementations, the Gen 3.1 TIL manufacturing process involves four operator interventions:

1. Tumor Fragment Isolation and Activation: On day 0 of the procedure, the tumor was dissected and final fragments of approximately 3 × ³ mm were generated (up to 240 fragments in total) and cultured in 1–4 G-REX100MCS culture flasks. Each flask contained up to 60 fragments, 500 mL of CM1 or DM1 medium, supplemented with 6,000 IU rhIL-2, 15 μg OKT3, and 2.5 × 10⁸ irradiated allogeneic mononuclear cells. The cultures were incubated at 37°C for 6–8 days.

2. TIL Culture Reactivation: On days 7-8, in both cases, replenish the culture by slowly adding CM2 or DM1 medium supplemented with 6,000 IU rhIL-2, 30 μg OKT3, and 5 × ^10⁸ irradiated allogeneic monocytes. Take care not to disturb the existing cells at the bottom of the culture flask. Incubate the culture at 37°C for 3-4 days.

3. Culture scaling up: Performed on days 10-11. During culture scaling up, in both cases, transfer all contents of the G-REX100MCS to a G-REX500MCS culture flask containing 4 L of CM4 or DM2 supplemented with 3,000 IU/mL IL-2. Incubate the culture flask at 37°C for 5-6 days until harvest.

4. Collection/Washing/Preparation: On days 16-17, reduce the volume of the culture flasks and combine them. Concentrate the cells and wash them with PlasmaLyte A pH 7.4 containing 1% HSA. Prepare the washed cell suspension with CryoStor10 at a 1:1 ratio and supplement with rhIL-2 to a final concentration of 300 IU/mL.

DPs are cryopreserved and stored in gaseous liquid nitrogen via controlled-rate freezing. *Complete standard TIL media 1, 2, or 4 (CM1, CM2, CM4) can be used instead of CTS ^™ OpTmizer ^™ T cell serum-free expansion medium, also known as the defining medium (DM1 or DM2), as mentioned above.

Procedure Description. On day 0, the tumor was washed three times and then fragmented into 3×3×3 final fragments. After fragmenting the entire tumor, the final fragments were then equally randomized and divided into three pools. One randomized fragment pool was introduced into each group, with the same number of fragments added according to the three experimental matrices.

Throughout the TIL amplification process, tumor L4063 was amplified using standard culture medium, and tumor L4064 was amplified using a specific culture medium (CTS OpTmizer). The components of the culture media are described herein.

CM1 complete medium 1: RPMI + glutamine, supplemented with 2mM GlutaMax ^™ , 10% human AB serum, gentamicin (50ug/mL), and 2-mercaptoethanol (55uM). The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM2 complete medium 2: 50% CM1 medium + 50% AIM-V medium. The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM4 complete medium 4: supplemented with AIM-V containing GlutaMax ^™ (2mM). The final medium formulation was supplemented with 3000 IU/mL IL-2.

The basal ^{medium for} T cell expansion is supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L).

DM1: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 6,000 IU/mL IL-2.

DM2: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 3,000 IU/mL IL-2.

All types of culture media used, i.e., complete (CM) and defined (DM) media, were prepared in advance and kept at 4°C until the day before use, and were preheated in an incubator at 37°C for up to 24 hours prior to the treatment day.

For both tumor types, TIL cultures were reactivated on day 7. For L4063, reactivation was performed on day 10, and for L4064, scale-up was performed on day 11. Both cultures were collected and cryopreserved on day 16.

Results achieved . Cell counts and survival percentages were determined during Gen 3.0 and Gen 3.1 procedures. Amplification under all conditions followed the details described in this example.

For each tumor, the fragments were divided into three equal pools. Due to the small size of the tumors, the maximum number of fragments per bottle could not be reached. Total viable cells and cell viability were assessed under each condition for three different procedures. Cell counts were determined as TVC at reactivation on day 7, TVC at scaling up on day 10 (L4064) or day 11 (L4063), and TVC at collection on day 16/17.

Cell counts on days 7 and 10/11 were considered as FIO. Folds of expansion were calculated by dividing the TVC at day 16/17 (when collected) by the TVC at day 7 (reactivation). To compare the three groups, the TVC at the collection day was divided by the number of fragments added to the culture on day 0 to calculate the average number of viable cells per fragment.

Cell counting and viability assays were performed on L4063 and L4064. On both tumors, the Gen3.1 assay produced more cells per fragment than the Gen 3.0 assay.

Total viable cell count and fold expansion; percentage of survival during the procedure. Percentage of survival under all conditions was obtained after reactivation, scaling up, and collection. Release criteria for survival percentage were compared to final TILs after collection on day 16/17. All conditions assessed exceeded the 70% survival criterion and were similar across all procedures and tumors.

Immunophenotyping analysis – Phenotypic characterization of TIL final products. Flow cytometry analysis of the final products was performed to test purity, differentiation, and memory markers. Under all conditions, the population percentages of TCRα/β, CD4+, and CD8+ cells were constant.

Extended phenotypic analysis of REP TILs was performed. In both tumors, TIL products showed a higher percentage of CD4+ cells under Gen 3.1 conditions compared to Gen 3.0, and a higher percentage of CD28+ cells from the CD8+ population under Gen 3.0 conditions compared to Gen 3.1 conditions under both conditions.

TILs collected from Gen 3.0 and Gen 3.1 processes showed phenotypic markers similar to CD27 and CD56 expression on CD4+ and CD8+ cells, and similar CD28 expression on CD4+-gated cell populations. Comparison of memory markers on TIL final products:

Frozen samples of TIL collected on day 16 were stained for analysis. The TIL memory state was similar in the Gen 3.0 and Gen 3.1 processes. Comparison of activation and depletion markers on the final TIL products:

The activation and depletion markers of gated Gen 3.0 and Gen 3.1 processes are similar in CD4+ and CD8+ cells.

Interferon-γ secretion after restimulation. For L4063 and L4064, collected TILs were restimulated overnight using coated anti-CD3 plates. In both tumors analyzed, higher IFNγ production from the Gen 3.1 process was observed compared to the Gen 3.0 process.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption levels under all conditions and processes, cell supernatants were collected and frozen at the start of reactivation on day 7, scale-up on day 10 (L4064)/day 11 (L4063), and collection on day 16/day 17. The supernatants were then thawed and analyzed. The amount of IL-2 in the cell culture supernatant was measured according to the manufacturer's protocol.

IL-2 consumption was similar throughout the entire Gen 3 and Gen 3.1 processes, evaluated under the same culture conditions. IL-2 concentrations (pg/mL) were analyzed in the used culture media collected from L4063 and L4064.

Metabolite analysis. For each condition, used culture supernatant was collected from L4063 and L4064 at day 7 for reactivation initiation, day 10 (L4064)/day 11 (L4063) for scaling up, and day 16/day 17 for collection. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

Compared to the complete medium (2 g/L), the glucose concentration in the determined medium was higher, at 4.5 g/L. Overall, the glucose concentration and consumption were similar for the Gen 3.0 and Gen 3.1 processes across each medium type.

An increase in lactate was observed, and the increase in lactate was similar under Gen 3.0 and Gen 3.1 conditions as well as in the two media (complete medium and deterministic medium) used for reactivation amplification.

In some cases, the standard basal medium contains 2 mM L-glutamine and is supplemented with 2 mM GlutaMax ^™ to compensate for the natural degradation of L-glutamine into L-glutamic acid and ammonia under culture conditions.

In some cases, the specified (serum-free) culture medium used does not contain L-glutamine compared to the basal medium and is supplemented only with GlutaMax ^™ at a final concentration of 2 mM. GlutaMax ^™ is a dipeptide of L-alanine and L-glutamine, which is more stable than L-glutamine in aqueous solution and does not spontaneously degrade into glutamate and ammonia. Instead, the dipeptide gradually dissociates into individual amino acids, thereby maintaining a low but sufficient concentration of L-glutamine to sustain stable cell growth.

In some cases, the concentrations of glutamine and GlutaMax ^™ decreased slightly on scale-up days but increased on collection days, reaching levels similar to or closer to those on reactivation days. For L4064, the concentrations of glutamine and GlutaMax ^™ showed a slight decrease at a similar rate under different conditions throughout the process.

The ammonia concentration was higher in the samples grown in the standard medium containing 2 mM glutamine + 2 mM GlutaMax ^™ compared to the samples grown in the defined medium containing 2 mM GlutaMax ^™ . Furthermore, as expected, a gradual increase or accumulation of ammonia was observed during cultivation. No difference in ammonia concentration was observed under the three different test conditions.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats on L4063 and L4064 cells during the Gen 3 and Gen 3.1 processes was measured using Flow-FISH technology. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Telomere measurements were performed. Telomere lengths in the samples were compared with those in the control cell line (1301 leukemia). The control cell line was a tetraploid cell line with long, stable telomeres that allowed for the calculation of relative telomere lengths. Similar telomere lengths were observed in the Gen 3 and Gen 3.1 processes evaluated in both tumors.

TCR Vβ lineage analysis

To determine the clonal diversity of cell products generated in each process, TIL end products were analyzed for clonal diversity analysis by sequencing the CDR3 portion of the T cell receptor.

Compare three parameters across three conditions:

● Diversity index of unique CDR3 (uCDR3)

● Total uCDR3 percentage

●For the first 80% of uCDR3:

○ Compare the total percentage of uCDR3 copies

○ Compare the frequency of unique clones

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 975 sequences, which is equivalent to 88% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 2163 sequences, which is equivalent to 87% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

The number of unique CD3 sequences identified from 1 × ^10⁶ cells collected on day 16 for different processes. Based on the number of unique peptide CDRs within the sample, the Gen 3.1 test conditions showed slightly higher clonal diversity compared to Gen 3.0.

The Shannon entropy diversity index is a reliable and commonly used comparative measure because the Gen 3.1 condition showed slightly higher diversity in both tumors compared to the Gen 3 process, indicating that the TCRVβ lineage of the Gen 3.1 test condition was more polyclonal than that of the Gen 3.0 process.

Furthermore, for tumors L4063 and L4064, the TCR Vβ lineage under the Gen 3.1 test conditions showed more than 87% overlap with the corresponding lineage of the Gen 3.0 process.

On the reactivation day, the IL-2 concentration on the used medium for the Gen 3.1 test of L4064 was lower than expected (similar to the Gen 3.1 control and Gen 3.0 conditions).

The low value may be attributed to pipetting error, but since the sample size was very small, it was impossible to repeat the measurement.

Conclusion . Compared with the Gen 3.0 and Gen 3.1 controls, the Gen 3.1 test conditions, which included feeder cells and OKT-3 on day 0, showed a higher TVC at day 16 collection. The TVC of the final product under the Gen 3.1 test conditions was approximately 2.5 times higher than that under Gen 3.0.

For the two tumor samples tested, the Gen 3.1 test conditions, with the addition of OKT-3 and feeder cells on day 0, reached the maximum capacity of the culture flasks at collection. Under these conditions, if a maximum of four flasks were started on day 0, the final cell dose could be between 80 and 100 × ^10⁹ TILs.

Maintain all quality attributes, such as phenotypic characterization, including the purity, depletion, activation, and memory markers of the final TIL product, between the Gen 3.1 test and the Gen 3.0 process.

In both tumor types analyzed, the final TIL product IFN-γ production in Gen 3.1 with added feeder cells and OKT-3 on day 0 was 3-fold higher than that in Gen 3.0, indicating that the Gen 3.1 process produces efficient TIL products.

No differences in glucose or lactate levels were observed under any of the test conditions. No differences in glutamine and ammonia were observed between the Gen 3.0 and Gen 3.1 processes under any of the culture medium conditions. Lower glutamine levels in the culture medium did not limit cell growth, indicating that the addition of GlutaMax ^™ alone to the culture medium is sufficient to provide the nutrients required for cell proliferation.

Scaling up was performed on days 11 and 10, respectively, and no significant difference was observed in the number of cells reached on the process's collection day. Furthermore, metabolite consumption was similar throughout the process in both cases. These observations suggest that the Gen 3.0 optimized process offers flexibility in terms of processing days, thereby promoting flexibility in manufacturing schedules.

As measured by CDR3 TCRab sequence analysis, the Gen 3.1 process, which added feeder cells and OKT-3 on day 0, showed higher clonal diversity compared to Gen 3.0.

Figure 32 illustrates the implementation scheme of the Gen 3 process (Gen 3 optimization process). Standard medium and serum-free CTS Optimizer medium can be used for TIL amplification in the Gen 3 optimization process. When using serum-free CTS Optimizer medium, it is recommended to increase the final concentration of GlutaMax ^™ on the medium to 4 mM.

Example 11: PD-1 gene knockout in TIL using TALEN

The embodiment described above illustrates the use of TALEN to generate PD-1 gene knockout (KO) TILs. In this embodiment, the general procedure is as follows:

Day 0 - Pre-REP

Day 7 - Activation

Day 12 - Electroporation

Day 13 - REP

Day 18 - Scale Expansion

Day 22 - Collection

Overview : This study used genetic engineering techniques to permanently knock out the PDCD-1 gene (PD-1 surface protein) using an mRNA construct. PD-1KO TILs were generated from one lung (L4256) and two melanoma (M1207 and M1209) tumors and then characterized.

Background : Binding between immune checkpoint receptors and their ligands has been shown to suppress T cell function. PD-1, a key inhibitory regulator expressed by activated/depleted T cells, has been extensively studied and is a major target for immunotherapy. Given the success of anti-PD-1 therapy in improving clinical outcomes in cancer patients, approaches targeting the PD-1/PD-L1 axis could enhance the efficiency of immunotherapy. Adoptive TIL therapy is a salvage immunotherapy for cancer, with an objective response rate of 56% in melanoma patients. Since PD-1 is regulated in TILs in response to antigen stimulation in vivo, the interaction between PD-1 and PD-L1 expressed by tumor cells may suppress TIL function. Gene editing of PD-1 has demonstrated high specificity for permanent gene disruption in human and mouse T cells. In the B16 mouse model, adoptive transfer of PD-1 T cells revealed superior tumor control and enhanced effector function and proliferative capacity of tumor-site infiltrating T cells. Therefore, this study examines whether PD-1 KO-mediated TILs can enhance TIL function in vivo after adoptive transfer.

Experimental design : Clinical manufacturing scale operations were performed as shown in the experimental layout in Figure 34, and an overview of the clinical manufacturing PD-1KO TIL process is provided in Tables 42 and 43.

The method is also applicable to the generation of PD-1/CTLA-4 dual-gene knockout TILs, PD-1/LAG-3 dual-gene knockout TILs, PD-1/CISH dual-gene knockout TILs, PD-1/TIGIT dual-gene knockout TILs, and PD-1/CBL-B dual-gene knockout TILs. In such cases, during the electroporation step, dual-gene knockout TILs can be generated by electroporating TILs with PD-1 and CTLA-4 TALEN mRNA, PD-1 and LAG-3 TALEN mRNA, PD-1 and CISH TALEN mRNA, PD-1 and TIGIT TALEN mRNA, or PD-1 and CBL-B TALEN mRNA, respectively.

Table 42: Outline of Pre-REP, Activation, Electroporation, and Resting Processes (Part I)

Table 43: Outline of REP, Scale-up, and Collection Process (Part II)

Results : Table 44 specifies the acceptance criteria used to evaluate the full-scale experimental efficacy, Table 45 specifies the additional end-product characterization tests performed, and Table 46 lists the tumors and related histories used in this study.

Table 44: In-process and Collected Product Release Testing and Acceptance Standards

Table 45: Characterization of the final product

Table 46: Tumors used in this study

On day 5, the total live cell count (TVC) after pre-REP yielded an average of 45 × ^10⁶ cells, with a mean survival rate of 73%. Cell doubling after activation was consistent across all three tumor types. Melanoma and the cervical indication both showed similar activation status, with activation markers of >50% CD25+ and CD71+. However, the lung tumor L4213 showed lower CD71 expression, at 33%. Ki67 expression was consistently greater than 90% in all three samples, indicating active cell proliferation. Table 47 summarizes the pre-REP and post-activation output for the three tumor types used in the study.

Table 47: Summary of Pre-REP and Post-Activation Cell Culture

parameter	Acceptance Standards	L4256	M1207	M1209
					Number of segments	≤100 segments	73	40	95
<![CDATA[TVC(×10<sup>6</sup>)]]>	≥5	26.9	13.4	69.2
					Survival rate (%)	N/A	97.9	88.6	97.5
<![CDATA[TVC(×10<sup>6</sup>)]]>	N/A	120.0	37.8	419.0
					Survival rate (%)	N/A	92.0	82.6	92.8
Double (D7 to D12)	N/A	2.2	1.5	2.6
					CD45+ / CD3+%	N/A	80.4	83.1	60.1
<![CDATA[CD25+%<sup>1</sup>]]>	N/A	92.2	88.5	66.8
					<![CDATA[CD71+%<sup>1</sup>]]>	N/A	75.6	87.8	48.9
<![CDATA[Ki-67+%<sup>2</sup>]]>	N/A	78.6	81.0	7.3

^1. Gating strategy of the group after activation on day 10: Lymphocytes > SSC single peak > FSC single peak > Live cells > CD3+CD45 > CD25+/CD71+

^2. Ki67 group's gating strategy: Lymphocytes > SSC unimodal > FSC unimodal > Live cells > CD45+ > Ki67+

On day 13 (after rest), survival rate and TVC recovery rate were calculated to determine the efficiency of electroporation. For all conditions, the recovery rate of the percentage of cells surviving after electroporation ranged from 82% to 92%. In electroporated samples, the TVC recovery rate ranged from 16% to 30%. Data from electroporation and resting are summarized in Table 48.

Table 48: Summary of Electroporation and REP Initiation

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The recovery rate is calculated by dividing the TVC or survival rate after electroporation by the TVC or survival rate before electroporation and then multiplying by 100.

^2. The TVCs injected into the REP were the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

The same number of cells were used to initiate the REP under both experimental and control conditions. As the REP scaled up, the average cell doubling occurred 4 times under all experimental and control conditions. At REP collection, TVC yield in the TriLink experimental group was similar to that in the simulated control group (TVC difference ≤20%). Non-electroporated TVC yield was slightly higher than that in the TriLink and simulated control groups. An average of 9.8 cell doublings was observed between day 11 and day 20.

The final product doses of the TriLink group were 16.5, 28.5, and 28.7 × ^10⁹ , respectively, with >90% cell viability and >98% CD45+CD3+ cell count. The final product was a highly enriched TIL product.

The data from the REP initiation and collection are summarized in Table 49.

Table 49: Post-REP Summary

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The TVC inoculated into the REP was the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

^2. The recovery rate is calculated by dividing the TVC after LOVO by the TVC before LOVO and multiplying by 100.

^3. Assuming all cells proceed to the hypothetical yield in both experimental and control conditions. On day 12 (before electroporation), cells are allocated for experimental (TriLink mRNA) and control conditions (simulated). Hypothetical yield is calculated by multiplying the collection by 3.

^4. Cell doubling is calculated using the formula “＝LOG(TVC on day 22 / TVC on day 11) /LOG(2)”.

^5. LOVO did not follow the plan.

^{The six} batches of materials were small in scale and did not undergo LOVO testing.

All TriLink mRNAs met the acceptance criteria.

Data on the gene knockout efficiency of PD-1KO TIL products evaluated are summarized in Table 50.

Table 50: TIL PD-1KO Summary

Data evaluating the TIL function of the collected samples (including IFNg, granzyme-B, and CD107a release output) are summarized in Table 51.

Table 51: Summary of TIL Functions

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. Stimulation gating targeting live cells/CD3+

The functionality of TILs was characterized based on overnight stimulation of the final product with anti-CD3/anti-CD28/anti-CD137 Dynabeads. Supernatants were collected and frozen 24 hours after stimulation. ELISA was performed to assess the concentrations of IFNg and granzyme B released into the supernatant. IFNg release was greater than 500 pg/mL, and all TIL cultures secreted high levels (15036 pg/mL–40185 pg/mL) of granzyme B after stimulation.

CD107a (LAMP-1) is a marker of lymphocyte degranulation and is associated with cytotoxic activity following antigen or polyclonal stimulation (Aktas E et al., 2009). Flow cytometry was used to analyze the cell surface expression of CD107a in response to PMA stimulation for 2 hours in both CD4 ⁺ and CD8 ⁺ cells. The results presented in Table 51 compare CD107A expression measurements with those under unstimulated conditions. Similar to the TIL products generated in previous studies, most of the TILs in the final product expressed CD107A upon stimulation with PMA/IO (CD4+ and CD8+ TILs).

Multicolor flow cytometry was used to characterize TIL purity, identity, and the memory subset of REP TILs. ≤1% of detectable B cells, monocytes, or NK cells were present in the final collected TILs (Table 11). REP TILs were primarily composed of TCRa/b cells with effector memory differentiation. The CD4/CD8 ratio varied between samples but was consistent across different conditions within a single sample. No significant differences were observed between experimental and control conditions.

Data showing TIL purity, identity, and memory phenotype are summarized in Table 52.

Table 52: Characterization of TIL purity, identity, and memory phenotype

Note: The gating strategy for TIL purity is as follows:

Monocytes: viable cells%, CD14+

NK (Natural Killer) cells: viable percentage, CD14-, CD3-, CD56+CD16+

B cells: viable percentage, CD14-, CD3-, CD19+

T-CM-Central Memory T Cells

T-EM-effect memory T cells

T-EFF/TEMRA-effect memory and RA+ effect memory

Data showing TIL activation and TIL depletion levels for CD4+, CD8+, and CD3+ are summarized in Tables 53-55.

Table 53: Activation and Depletion States of CD4+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD4+ > CD4+ frequency %

Table 54: Activation and Exhaustion States of CD8+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD8+ > CD8+ frequency %

The CD4 and CD8 differentiation, activation, and depletion states under the TriLink mRNA condition were comparable to those under the control condition.

Table 55: Activation and Depletion States of CD3+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD3+ frequency %

The activation and depletion status of TriLink mRNA in the CD3+ population were comparable to those in the control population.

Conclusion : The PD-1KO TIL process was developed at clinical manufacturing scale to amplify PD-1KO TIL to >16e9 within 20 days. All three batches of TriLink mRNA used at development scale met the acceptance criteria for release parameters.

All quality properties of TILs generated using the PD-1-KO TIL process, such as phenotypic characterization (including purity, memory, activation, exhaustion markers, and function), are comparable to those of melanoma Gen 2. See Table 56.

In summary, this work demonstrates that greater than 50% PD-1KO efficiency can be achieved in the clinical manufacturing of PD-1KO TILs using TriLink mRNA, thus supporting the use of TriLink mRNA in clinical manufacturing.

Table 56: Summary Table

The alternative PD-1 gene knockout (KO) TIL process is shown in Figure 35 and Table 57.

Table 57: Overview of the alternative full-scale process for PD-1 TIL culture

Example 12: Preclinical activity and manufacturing feasibility of gene-modified PDCD-1 gene knockout (KO) tumor-infiltrating lymphocyte (TIL) cell therapy

background

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TILs; lifileucel, LN-145) has demonstrated encouraging efficacy and safety in both ICI-injected and ICI-naïve settings in patients with advanced solid tumors. A single-dose lifileucel TIL cell therapy achieved a durable response in a post-ICI setting in patients with advanced/unresectable melanoma (Sarnaik AA, Hamid A, Khushalani NI et al., J Clin Oncol. 2021; 39(24):2656-2666; Larkin JMG, Sarnaik A, Chesney JA et al., J Clin Oncol. 2021; 39(Supplement 15): Abstract 9505.), with an objective response rate (ORR) of 36%, and the duration of response (DOR) was not reached after 33.1 months of follow-up. In patients with advanced melanoma who have not received ICI treatment, early combination therapy with lifileucel and pembrolizumab resulted in an ORR of 60% and a complete response rate of 30% (O'Malley D, Lee S, Psyrri A et al., J Immunother Cancer. 2021; 9(Supplement 2): Abstract 492).

Despite the effectiveness of anti-programmed cell death protein (PD)-1 ICI therapy, it is limited by poor tumor penetration, internalization, and endocytic clearance (Thurber GM, Schmidt MM, Dane Wittrup K. Adv Drug Deliv Rev. 2008; 60(12):1421-1434; Less JR, Posner MC, Boucher Y, Borochovitz D, Wolmark N, Jain RK. Cancer Res. 1992; 52(22):6371-6374; Netti PA, Baxter LT, Boucher Y, Skalak R, Jain RK. Cancer Res. 1995: 55(22): 5451-5458; Bordeau BM, Balthasar JP. Cancer Biol Med. 2021; 18(3): 649-664; Saga T, Neumann RD, Heya T et al., Proc Natl Acad Sci U SA. 1995; 92(19): 8999-9003); In contrast, TILs are actively transported into the tumor, can move independently by chemitraction, and are not cleared or internalized (Restifo NP, Dudley ME, Rosenberg SA. Nat Rev Immunol. 2018; 12(4): 269-281). Administration of anti-PD-1 antibodies is associated with immune-related adverse events (AEs) due to nonspecific upregulation of immune pathways via systemic activation and proliferation of T cells (Postow MA, Sildow R, Hellmann MD. N Engl J Med. 2018; 378:158-168).

IOV-4001 is a PDCD-1 gene knockout (KO) TIL cell therapy that enhances the efficacy of TIL cell therapy and eliminates the need for systemic anti-PD-1 therapy, while avoiding short-term and long-term systemic adverse events associated with anti-PD-1/PD-L1 therapy. Transcription activator-like effector endonucleases (TALENs) are hybrid molecules composed of a DNA-binding domain and a FokI nuclease. Combination of the two TALEN arms of the PDCD-1 gene encoding PD-1 mediates DNA double-strand breaks, leading to gene damage and PD-1 inactivation (Gautron AS, Juillerat A, Guyot V et al., Mol Ther Nucleic Acids. 2017; 9:312-321; Menger L, Sledzinska A, Bergerhoff K et al., Cancer Res. 2016; 76:2087-2093; Qasim W, Zhan H, Samarasinghe S et al., Sci Transl Med. 2017; 9(374):eaaj2013). A process has been established to generate TALEN-mediated PDCD-1KO TILs and amplify them to a treatment-relevant number with potent effector function and phenotypic markers indicating functional TILs (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; Sep 19–21, 2020. Abstract 1052p).

This embodiment describes the (1) preclinical activity of IOV-4001 in vivo, (2) clinical-scale manufacturing process development, and (3) phenotypic and functional characterization.

method

In summary, hIL-2NOG mice transplanted with melanoma tumor cells received autologous PD-1KO TILs (generated using an mRNA construct), TIL-mimicking (electroporation without TALEN), TIL-mimicking plus anti-PD-1 antibody adoptive transfer, or no adoptive transfer (n=14 each). Tumor size was measured twice weekly for 39 days. A 22-day clinical-scale manufacturing process was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, for the generation of PD-1KO TILs. The final PD-1KO TIL product was characterized by total live cells (TVC), purity (survival percentage), identity (CD45 ⁺ CD3 ⁺ %), and function (PD-1KO efficiency).

manufacture :

A 22-day clinical-scale manufacturing process for IOV-4001 was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, to generate PDCD-1KO TILs (using an mRNA construct). Development operations were performed at Iovance Process Development (Tampa, FL) on a non-Good Manufacturing Practice (GMP) scale. Manufacturing operations were performed at a contract manufacturing organization (CMO) on a GMP scale.

In vivo antitumor activity :

Mice expressing human interleukin-2 (hIL2) under the control of the cytomegalovirus promoter (hIL-2NOG)15 were transplanted with melanoma tumor cells and received (A) autologous PDCD-1KO TIL, (B) TIL mimicry (TIL electroporation without TALEN), (C) TIL mimicry plus anti-PD-1 antibody, or (D) TIL-free adoptive transfer (n=14 each). Tumor size was measured twice a week for 39 days.

Product release :

The final PDCD-1KO TIL product was characterized as follows:

●Total viable cells (TVC) and purity (percentage of viable cells) were determined using an NC-200 ^™ (ChemoMetec, Denmark) automated cell counterstaining system via acridine orange/4',6-diamidindo-2-phenylindole (DAPI) counterstaining.

● Identity (CD45+CD3+ phenotype), analyzed by immunofluorescence staining and flow cytometry.

●Efficacy (interferon-γ [IFNγ] release), measured by ELISA using an IFNγ ELISA kit (R&D Systems, Minneapolis, MN, USA).

PDCD-1KO efficiency was evaluated by flow cytometry based on PD-1 expression in PDCD-1KO TILs compared with mimic TILs.

Phenotype :

Two multicolor flow cytometry teams were used to determine extended phenotypic markers of the final collected PDCD-1KO TIL products to characterize TIL purity, identity, memory subsets, activation, and depletion states.

Characterization :

IL-2 assay: To assess the safety of the PDCD-1KO TIL product, the in vitro proliferation capacity of the final product in the absence of IL-2 was evaluated over a 28-day period.

Karyotype determination: Cytogenetic examination was performed by NeoGenomics Laboratories (Fort Myers, FL, USA). Briefly, cryopreserved PDCD-1KO TIL samples were quiescent and activated to collect metaphase cells for G-banding cytogenetic analysis. Three replicates of mitotic cells were analyzed, fixed, and stained for G-banding.

Statistical analysis :

Unpaired Student's t-test was used to analyze phenotypic differences, and Wilcoxon's rank-sum test was used to detect differences in in vivo studies in mice; p < 0.05 was considered statistically significant.

result

In summary, for mice treated with PD-1KO TIL (6±2.8), mean ± SEM tumor size ( ^mm² ) at day 39 showed superior tumor control compared to simulated TIL (26±8.5, P<0.05), simulated TIL + anti-PD-1 (33±8.8, P<0.01), and no adoptive TIL metastasis (112±8.4, P<0.0001). Product properties from six clinical-scale manufacturing operations of PD-1KO TIL were acceptable, with median (range) TVC, purity, and identity of 8.3 × ^10⁹ (0.9 × ^10⁹ –35.7 × ^10⁹ ), 94% (91%–99%), and 99% (98%–99%), respectively. The median (range) PD-1KO efficiency was 48% (31%–84%). PD-1KO TILs exhibit comparable functions and phenotypes (differentiation, memory, activation, and exhaustion) to those of analog TILs.

As shown in Figure 38, enhanced in vivo antitumor activity was observed in mice treated with PDCD-1KO TIL compared to mice treated with TIL-mimicking alone or TIL-mimicking alone plus anti-PD-1 antibody.

As shown in Figure 39, all PDCD-1KO TIL products met the release criteria for dose, purity, identity, potency, and PDCD-1KO efficiency. No statistically significant differences were observed between the development and manufacturing processes. Both development and manufacturing processes produced final PDCD-1KO TIL products with comparable doses (A) and survival rates (B). The median (range) identity (CD45+CD3+%) of the final TIL products in the development and manufacturing processes was 98.5% (98%–100%) and 98.7% (96%–99%), respectively (C). The median IFNγ release in the development and manufacturing processes of the final PDCD-1KO TIL products was 4015 pg/mL and 4725 pg/mL, respectively (D). The median (range) PDCD-1KO efficiencies in the development and manufacturing processes were 63% (48%–81%) and 62% (31%–91%), respectively (E). The PDCD-1KO TIL product is comparable to the simulated TIL in terms of growth, purity, identity, and potency. As previously shown (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; September 19–21, 2020. Abstract 1052p), the dose, purity, identity, and potency results between the simulated and PDCD-1KO were comparable in development operations (data not shown).

As shown in Figure 40, the CD28 biomarker was highly expressed in both PDCD-1KO and simulated TILs in the development and manufacturing samples, while other biomarkers such as CD27, CD57, and KLRG1 were expressed at low levels (A). Subsets of naive (TN), central memory (TCM), effector memory (TEM), and effector memory RA+ (TEMRA) T cells were defined using CD45RA and CCR7 expression. Most TIL batches primarily exhibited the effector memory phenotype (B). No statistically significant differences in TIL differentiation biomarkers or memory phenotypes were observed between PDCD-1KO and simulated TILs during the development and manufacturing operations.

As shown in Figure 41, multicolor flow cytometry was used to characterize TIL activation and inhibitory receptor expression on CD4+ (A) and CD8+ TILs (B). No statistically significant differences in biomarker expression were observed between PDCD-1KO and simulated TILs during development and manufacturing operations.

As shown in Figure 42, the IL-2-independent proliferation assay demonstrated that no PDCD-1KO TIL products underwent malignant transformation after TALEN-mediated genome editing. In the absence of IL-2 (A and B), neither stimulated (anti-CD3/CD28) nor unstimulated samples proliferated. All samples cultured in the presence of IL-2 showed proliferation (A and B).

The karyotype analysis results from the PDCD-1KO TIL products are summarized in Figure 43. No clonal chromosomal abnormalities were observed in the G-banding analysis, indicating no genotoxicity following TALEN-mediated PDCD-1 genome editing. All samples analyzed by G-banding generated sufficient metaphase cells for a comprehensive study; normal G-banding patterns were observed.

in conclusion

In the presence or absence of anti-PD-1, PDCD-1KO TILs exhibited superior in vivo antitumor activity compared to mimic TILs (electroporation without TALEN), indicating that endogenous PD-1 inhibition can confer a functional advantage over antibody combinations. Clinical-scale manufacturing of PDCD-1KO TILs is feasible, and the quality properties and phenotype of the TIL products are acceptable. TIL properties were comparable across all development and manufacturing operations and met product release criteria. As determined by IL-2-independent proliferation assays, no malignant transformation occurred in PDCD-1KO TIL products following TALEN-mediated genome editing. No TALEN-induced clonal chromosomal abnormalities were identified by G-banding. Importantly, the absence of intact PDCD-1KO in the TIL products preserved other PD-1-dependent in vivo cellular functions. In conclusion, these data support the clinical investigation of IOV-4001 (an autologous PDCD-1KO TIL cell therapy) as a potential treatment option for patients with advanced solid tumors.

Example 13: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated, electroporated, and subjected to an 11-day REP process.

Pre-REP cells were stimulated for two days with plate-bound OKT3 (300 ng/ml) or TransAct (1:100), followed by electroporation of 5e6 cells resuspended in an electroporation buffer called “T buffer” in 4 mm spaced electroporation reaction cuvettes, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

After electroporation, cells were allowed to rest overnight at 30°C in CM1 medium containing IL-2, followed by REP. Simulated cells were seeded at different initial numbers (100kJ, 50kJ, 20kJ, 10kJ) and tests were performed to compare gene knockout efficiency.

result

Figures 44A and 44B show cell viability and recovery folds before electroporation. Specifically, Figure 44A shows cell viability after thawing compared to 2 days after stimulation with OKT3 or TransAct, and Figure 44B shows the recovery folds after thawing and stimulation with OKT3 or TransAct.

Figures 45A and 45B show the cell viability and recovery fold of cells after electroporation. Specifically, Figure 45A shows the recovery fold of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C, and Figure 45B shows the cell viability of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C.

Figures 46A-46C show that, in the case of CD3+, CD8+, and CD4+ cells, TILs stimulated by TransAct exhibited lower KO efficiency compared to TILs stimulated by OKT3.

Example 14: Comparison of overnight rest at 30°C and 37°C

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated and electroporated.

Pre-REP cells were stimulated with TransAct (1:100) for two days, followed by electroporation of 5e6 cells resuspended in T buffer in electroporation reaction cups with a 4 mm spacing, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

Following electroporation, cells were seeded in equal numbers into 96-well plates and allowed to rest overnight at 30°C or 37°C in CM1 medium containing IL-2 or IL-15. Cells were then counted to determine the fold resuscitation and viability.

result

Figures 47A and 47B show cell viability and recovery folds after electroporation under all conditions (IL-2 or IL-15). Specifically, Figure 47A shows the recovery folds of cells after overnight rest at 30°C or 37°C, and Figure 47B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 48A and 48B show the cell viability and fold recovery rate of cells after electroporation when 6000 IU/mL IL-2 was used. Specifically, Figure 48A shows the fold recovery rate of cells after overnight rest at 30°C or 37°C, and Figure 48B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 49A and 49B show cell viability and recovery folds after electroporation under various conditions. Specifically, Figure 49A shows the recovery folds of cells after overnight rest at 30°C or 37°C when using various concentrations of IL-2 or IL-15, and Figure 49B shows the cell viability after overnight rest at 30°C or 37°C when using different concentrations of IL-2 or IL-15.

Gene knockout efficiency was evaluated as described in Example 13. TILs were stimulated overnight with anti-CD3/CD28 beads at a ratio of 1 bead to 5 cells. Figures 50A-50C show that a decrease in KO efficiency was observed at the recommended 1:100 dilution under non-GMP TransAct stimulation.

Example 15: Comparison of TALEN mRNA concentration and incubation conditions

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed, activated, electroporated and subjected to a 10-day REP process.

Pre-REP cells were stimulated with OKT3 (300 ng/mL plate-bound) for two days, followed by electroporation of 1e6 cells resuspended in T buffer in electroporation reaction cuvettes with a 1 mm spacing. The right arm or left arm was given 4 μg, 2 μg, 1 μg, and 0.5 μg of PD-1 TALEN per 1e6 cells, respectively.

After electroporation, cells were seeded in the same number in 48-well plates and allowed to rest overnight in CM1 medium containing IL-2 at 30°C or 37°C, followed by REP.

result

Figures 51 and 52 show the cell viability and fold recovery rate of cells after electroporation with different concentrations of PD-1 TALEN mRNA. Specifically, Figure 51 shows the cell viability after electroporation with different concentrations of PD-1 TALEN mRNA, and Figure 52 shows the fold recovery rate after electroporation with different concentrations of PD-1 TALEN mRNA. No significant changes in viability or recovery rate were observed after electroporation with different concentrations of PD-1 TALEN mRNA.

Figures 53A and 53B show the cell viability and fold recovery rate when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C. Specifically, Figure 53A shows the fold recovery rate of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C, and Figure 53B shows the cell viability of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C. No significant changes in viability or recovery rate were observed when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C.

Gene knockout efficiency was evaluated as described in Example 13. Figures 54A-54C show the PD-1 KO efficiency in CD3+, CD8+, and CD4+ cells. The optimal KO efficiency of PD-1 TALEN was observed at 4 μg mRNA/million cells and overnight rest at 30°C. This concentration can be target-specific and also depends on the cell concentration used during electroporation. The concentration of TALEN mRNA used during the electroporation process did not appear to affect recovery rate and survival.

Example 16: Comparison of washing steps and centrifugation speed

method

Pre-REP TIL lines (N=4) from different indications (head and neck and lung) were thawed, activated (OKT3 plate binding 300 ng/mL), and subjected to washing steps that are usually performed before electroporation.

Pre-REP cells were stimulated for two days and then aliquoted into individual tubes at 5e6 cells per tube. They were washed with PBS, precipitated, washed with cell perforation buffer, precipitated again, and then resuspended in cell perforation buffer. Cells were resuspended in 500 μL of cell perforation buffer and counted by diluting the cells 1:10 in CM1. Cell counting was performed after each rotation to determine the percentage of cells lost during the washing step.

Repeat the procedure using different centrifugation speeds (400g, 300g, 200g, for 5 minutes) to improve cell recovery rate.

result

Figures 55A and 55B show the cell number (Figure 55A) and viability (Figure 55B) after various washing steps involving centrifugation at 400g for 5 minutes and rotation 3 times.

Figures 56A and 56B show the number of cells after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 57A and 57B show cell viability after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 58A and 58B show the total number of cells and the total survival rate of cells after various rotation conditions, and Figure 59 shows the percentage of cells lost after various rotation conditions.

The results showed that washing with cell perforation buffer led to cell loss, and that the recovery rate may have been improved when cells were centrifuged in cell perforation buffer at 300g instead of 400g. Therefore, a single cell perforation buffer washing step is preferred.

Example 17: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates containing 300 ng/mL, TransAct 1:10 and TransAct 1:17.5, and then electroporated with PD-1 TAL.

result

Figures 60A-60C show the percentage loss and survival during electroporation using various stimulation methods, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation (Figure 60C). The results indicate that TransAct stimulation exhibits better recovery and survival rates after electroporation compared to OKT3.

Figures 61A-61C show the PD-1 gene knockout efficiency in CD3+ (Figure 61A), CD4+ (Figure 61B), and CD8+ (Figure 61C) cells using various stimulation methods. The results indicate that activation with OKT3 and TransAct leads to similar KO efficiency.

Figures 62A-62B show the cell viability (Figure 62A) and fold expansion (Figure 62B) of REP collections using various stimulation methods.

Example 18: Comparison of incubation temperatures after electroporation

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates at 300 ng/mL, and then electroporated with PD-1 TAL.

After electroporation, cells were incubated overnight at different temperatures (25, 28, 30, 32, 35, 37 °C) and REP was performed the following day. Cell loss and viability, as well as PD-1 gene knockout efficiency, were evaluated after overnight incubation.

result

Figures 63A-63B show the percentage of cell loss (Figure 63A) and cell viability (Figure 63B) after electroporation at different incubation temperatures.

Figures 64A-64C show the gene knockout efficiency in CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells using different incubation temperatures.

Figures 65A-65B show the fold expansion (Figure 65A) and cell viability (Figure 65B) of REP collections at different incubation temperatures.

Example 19: Comparison of Stimulus Day Timing Selection

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated with GMP TransAct 1:17.5 on different dates (days 0, 3, 5, and 7).

Electroporation of cells with PD-1 TAL was performed on days 9 and 12 to determine the optimal dates for stimulating pre-REP cells to maximize PD-1 KO efficiency and pre-REP cell number.

result

Figures 66A-66C show cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C) of cells stimulated on different dates.

Figure 67 shows the percentage growth of cells stimulated on different dates at 3 days of rest.

Example 20: Overview of PD-1KO TIL Products

"PD-1KO TIL Product" is an autologous tumor-infiltrating lymphocyte (TIL) preparation. These lymphocytes have been genome-edited with a transcription activator-like effector nuclease (TALEN) to disrupt the gene for programmed cell death protein-1 (PD-1). The PD-1 TALEN is engineered to bind to a distinct DNA sequence adjacent to the nucleotide lysis target site on the PDCD1 gene. DNA binding leads to the dimerization of two separate FokI endonuclease domains, producing a functional DNA endonuclease that can generate a sequence-specific double-strand break (DSB) in exon 2 (the central protein-coding region of PDCD1). This DSB is repaired via endogenous error-prone DNA repair pathways (e.g., non-homologous end joining (NHEJ)), resulting in nucleotide insertions or deletions, thereby disrupting the protein-coding sequence of PD-1.

The 4-step PD-1KO TIL product manufacturing process is a modified version of the method used to produce the autologous TIL product lifileucel (IND 16317 and 16819). This process consists of the following steps: a first step of TIL growth in the presence of IL-2, followed by transient activation of T cells with TransAct (a bead-free colloidal polymer nanomatrix conjugated with humanized CD3 and CD28 agonists), electroporation of PD-1KO TALEN mRNA (left and right arms), overnight rest at 30°C, and a final rapid expansion protocol (REP) step.

Cross-study PD-1 KO TIL product characterization data (Table 58) and process development data indicate that changes in the manufacturing process do not affect product quality (characteristics, survival rate, PD-1 gene knockout efficiency) and result in an average PD-1 KO efficiency of approximately 60%. In the PDX mouse model system, its antitumor activity is superior to that of the simulated control TIL in the presence of pembrolizumab.

Table 58. List of Non-Clinical Studies

Abbreviations: GLP = Good Laboratory Practice; IL-2 = Interleukin-2; KO = Gene Knockout; OCA = Oligonucleotide Capture Assay; PD-1 = Programmed Cell Death Protein-1; PDCD1 = Gene of Programmed Cell Death Protein-1; PDX = Patient-Derived Xenograft; TALEN = Transcription Activator-like Effector Nuclease; TCR = T Cell Receptor; TIL = Tumor-Infiltrating Lymphocytes

¹ This study evaluates different batches of PD-1KO TIL products, some of which were evaluated as part of a GLP compliance study.

Overview

The non-clinical studies conducted support the safety of the PD-1KO TIL product initiating clinical studies through the following:

● Confirmed that PD-1 TALEN expression in TILs is transient after electroporation;

● Characterization using NGS confirmed that 19 of the top 20 potential off-target sites identified in the OCA analysis were free of TALEN-related mutagenesis. Minimal off-target activity was detected at one candidate site (Cand 3). This is unlikely to pose a safety risk to the product because of the low mutation frequency and the fact that the site corresponds to a non-coding RNA locus not known to be associated with the disease.

● No clonal chromosomal aberrations were identified in the karyotype analysis of the PD-1KO TIL drug product, therefore there is no evidence that TALEN-mediated genome editing has significant genotoxicity;

● No evidence of IL-2-independent proliferation was found, thus demonstrating that no malignant transformation was observed in any of the PD-1KO TIL drug products tested.

Furthermore, non-clinical studies support the potential antitumor activity and mechanism of action of PD-1KO TIL products based on the following observations:

● Reproducible high levels of TALEN-mediated PDCD1 mutagenesis (approximately 60% on average) were achieved in the genome of autologous TILs, with the PDCD1 mutagenesis rate correlated with decreased PD-1 expression. Therefore, PD-1KO TIL products constitute true PD-1KO gene therapy products.

● The general characteristics of autologous TILs, which are considered crucial for anti-tumor activity, have been shown to be unaffected or positively affected by PD-1KO, including survival, proliferative capacity, differentiation status, memory T cell subset, activation/exhaustion status, TCR Vβ diversity, T cell effector function, and cell lysis activity.

● Enhanced tumor burden control was observed in PDX mouse models treated with ACT and PD-1KO TIL products, compared with control groups that did not use ACT, ACT with unmodified autologous TIL, and ACT with unmodified autologous TIL combined with anti-PD-1 antibody.

In summary, these studies demonstrate that isolating, expanding, and genetically engineering PD-1 knockout T cells from various tumors is safe and exhibits potent antitumor activity in both in vitro and in vivo models. These observations suggest that PD-1KOTIL products possess the potential and manageable safety profile of an autoimmune therapy for treating patient populations constituting unmet medical needs.

result

Figures 68A-68C show the PD-1 gene knockout efficiency. Figure 68A shows the PD-1 KO efficiency determined by measuring PD-1 receptor expression on the PD-1 KO TIL product using flow cytometry. Figure 68B shows the PD-1 KO efficiency determined by measuring the total number of PDCD1 genes with insertions/deletions using NGS. Figure 68C shows the correlation between the PD-1 KO efficiency assessed by flow cytometry and the PDCD1 gene modifications determined using NGS.

Figure 69 shows that most of the insertions/deletions in PDCD1 after TALEN genome editing were nucleotide deletions.

Phenotypic characterization

The proliferative capacity, survival, and immunophenotype of PD-1KO TIL products relative to unmodified autologous TILs were evaluated using a study-scale process derived from five different types of tumors (breast (n=5), lung (n=2), HNSCC (n=2), melanoma (n=2), and ovary (n=1)).

Cell viability and proliferation capacity

The PD-1KO TIL product exhibited cell viability comparable to non-electroporated (NE) TILs, with an average viability of approximately 93% for both types of samples. The proliferative capacity of the PD-1KO TIL product was also comparable to that of NE TILs, with average fold increases of 1217 and 1565, respectively, during the REP step of the production process.

Differentiation status of PD-1KO TIL products and NE TIL

PD-1KO TILs exhibit a differentiation state comparable to NE TILs, as determined by similar expression levels of CD27, CD28, CD56, BTLA, and KLRG-1. Therefore, PD-1 inactivation does not alter the differentiation state of TILs.

Memory T cell subsets in PD-1KO TIL products and NE TIL

In this study, over 98% of PD-1KO TIL products and NE TILs were T effector cells (CD45RA-CCR7-), while _{the TCM} subset (CD45RA-CCR7+) accounted for less than 1% of the cells analyzed. Importantly, the proportion of terminally differentiated T cells was negligible.

Activation and depletion states in PD-1KO TIL products and NE TIL

As indicated by the expression levels of CD69, CD25, and DNAM, the activation state of the PD-1KO TIL product was relatively high and comparable to that of NE TIL. Consistent with its recent activation, both the PD-1KO TIL product and NE TIL expressed high levels of co-inhibitory molecules (TIGIT and TIM-3). These results suggest that the activation and depletion states are not altered by PD-1 inactivation.

In summary, PD-1 gene knockout did not significantly affect TIL survival, proliferation capacity, or immune phenotype, indicating that the activity of PD-1KO TIL products associated with these biological characteristics was similar to that observed in unmodified autologous TILs.

Figures 70A-70B show the distribution of TCR Vβ isotypes in the CD3+PD-1- subset within the main PD-1KO TIL products and NE TILs. Main TILs generated from lung tumor L4022 (Figure 70A) and breast tumor EP11017 (Figure 70B) were electroporated with PD-1 TALEN mRNA (PD-1KO) or not electroporated (NE). Twenty-four TCR Vβ families were evaluated by flow cytometry in the CD3+PD-1-T cell subset. The percentage of TCR Vβ isotypes is plotted in pie charts. All remaining isotypes not identified by flow cytometry are pooled and shown in light blue (other). The color represents each TCR Vβ isotype, as indicated in the legend below the pie chart.

Figures 71A-71B show the effector function of PD-1KO TILs as measured by MLR and multifunctionality. For Figure 71A, TILs were generated in duplicate from melanoma (n=3), breast cancer (n=1), and lung cancer (n=1) samples. The responsiveness of NE TILs (black circles) and PD-1KO TILs (white circles) to HLA-mismatched lymphocytes was tested. IFN-γ secretion from the supernatant was assessed using ELISA. The horizontal and vertical bars represent the mean and standard error, respectively. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance. For Figure 71B, TILs were generated from melanoma (n=1) and breast cancer (n=3) samples. The multifunctionality intensity index (PSI), a measure of the ability to produce multiple cytokines at the single-cell level, was measured in activated host PD-1KO and NE TILs. The results are shown as a bar graph of the mean and corresponding standard error of cytokines involved in the inflammatory (pink), regulatory (beige), chemoattractive (purple), stimulative (blue), and effector (green) pathways. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance.

In vivo studies: Patient-derived xenograft (PDX) mouse model studies

This study compared the antitumor activity of PD-1KO TIL products with unmodified TILs in a patient-derived xenograft (PDX) model grown in hIL-2 transgenic mice. Paired autologous PDX tumor cell lines/TIL formulations derived from freshly excised human melanoma lesions (n=1; M1152) were used in this study. The PD-1KO efficiency level was 75%. No differences in cell viability, proliferation, differentiation, memory T cell subsets, or exhaustion status were observed between M1152 PD-1KO and simulated TILs.

Characterization of PDX-derived melanoma tumor cell lines

The M1152 PDX tumor cell line is a PDX tumor-derived gene from NSG mice implanted with melanoma tumor fragments. The PDX melanoma cell line was characterized by measuring melanoma tumor-specific markers called melanoma-associated chondroitin sulfate proteoglycan (MCSP) and PD-L1 expression via flow cytometry. Approximately 80% of the tumor cells expressed MCSP protein, indicating that the majority of these cells were of melanoma origin. IFN-γ treatment induced an upregulation of PD-L1 from 13% to 82% within 48 hours. This encapsulates an in vivo drug resistance mechanism in which tumor cells increase PD-L1 expression in response to T cell secretion of IFN-γ during tumor cell lysis and supports the relevance of the M1152 PDX model for assessing M1152 PD-1 KO TIL activity.

In vivo tumor burden control of M1152 PD-1KO TIL

To determine the effect of PD-1KO TILs on tumor growth, PD-1KO or TIL-mimicking cells were adoptively transferred into hIL-2NOG mice implanted with autologous melanoma cells. Combinations of anti-PD-1 and TIL-mimicking cells served as controls for PD-1/PD-L1 pathway blockade. The in vivo efficacy of PD-1KO TILs was assessed by tumor burden reduction.

Figure 72 shows the in vivo antitumor activity of the M1152 PD-1KO TIL product. hIL-2NOG mice (n=14 per treatment group) transplanted with melanoma tumor cells underwent adoptive metastasis with either PD-1KO or a TIL-mimicking agent. Anti-PD-1 antibody treatment in combination with a TIL-mimicking agent was included as a control for PD-1/PD-L1 blockade. Tumor volume was calculated as length × width and plotted as a function of time post-implantation. Statistical significance was assessed using the Wilcoxon rank-sum test, with *, **, and **** indicating p-values <0.05, <0.01, and <0.0001, respectively, and considered statistically significant.

Tumor reduction was observed in mice treated with PD-1KO and TIL-mimicking antibodies, with or without anti-PD-1 antibody therapy. This indicates that TILs can recognize and lyse transplanted tumor cells, thus validating the tumor-specific recognition of TILs via MHC and TCR conjugation. Furthermore, even in combination with anti-PD-1 therapy, PD-1KO TILs induced superior tumor control relative to TIL-mimicking TILs. The failure of anti-PD-1 to enhance the in vivo efficacy of TIL-mimicking TILs in immunodeficiency and the lack of primary and secondary lymphoid organs in hIL-2NOG mice suggests that the in vivo mechanism of systemic PD-1 suppression may require an intact immune system. Conversely, the improved antitumor activity of PD-1KO TILs compared to TIL-mimicking TILs indicates that endogenous PD-1 suppression confers a functional advantage on TILs.

Therefore, this study demonstrates that TALEN-mediated PD-1KO confers a functional advantage on TILs. This finding is consistent with several published studies that describe an enhanced ability of PD-1KO T cells to control tumor growth relative to unmodified T cells (Menger 2016, Marotte 2020).

Figures 73A-73B show the persistence of TALEN protein in autologous TILs as a function of time, as measured by Western blotting. TILs generated from melanoma (n=6; M1011, M1017, M1025, M1030, M1034, M1040) were electroporated with PD-1 TALEN mRNA. NE TILs were used as a baseline control. TALEN protein was detected in whole-cell extracts from TILs by Western blotting at 8, 10, 12, 14, and 18 hours post-electroporation (Figure 73A) and on days 1, 2, 4, and 6 (Figure 73B). The intensity of the TALEN-specific bands was quantified by densitometric analysis, with the resulting OD values averaged over time and background corrected for the mean values of NE TILs. The mean values are plotted as bars, and the standard error is shown as a vertical line.

Quantification of mutagenesis at target sites and candidate off-target sites in PD-1 TALEN

Information about each candidate off-target site is included in Table 59.

Table 59. Off-target site analysis

IL-2-independent proliferation assay

By day 28 of analysis, none of the PD-1KO TIL product samples evaluated using CD3/CD28 activation proliferated in the absence of IL-2. All samples cultured in the presence of IL-2 proliferated by day 28, with a minimum amplification of 1.7-fold and a maximum of 407-fold. Notably, paired TALEN-edited TILs and unmodified TILs isolated from the same tumor showed comparable overall amplification folds across samples. These results demonstrate the safety of the PD-1KO TIL drug product by confirming the absence of cell proliferation in the absence of IL-2, indicating no malignant transformation of cells in this product.

Cytogenetics/karyotype analysis

Two aberrant karyotypes were observed, with X chromosome monosomy detected in 4–5 of the 20 metaphase diffuses analyzed. These structural abnormalities were detected in both non-genome-edited TIL samples and TALEN-edited TIL samples, therefore, these abnormalities cannot be attributed to TALEN-mediated genome editing. Furthermore, as assessed in a cytogenetic analysis of 200 metaphase cells, no significant increase in mutagenesis was observed in PD-1 TALEN-edited TILs compared to unmodified TILs. Based on these results, it is concluded that no evidence of TALEN-induced clonal chromosomal aberrations was found in any of the tested samples, and therefore, no significant genotoxicity was identified following TALEN-mediated genome editing.

Example 21: Exemplary TIL Manufacturing Process

Figures 74A-F depict an exemplary TIL manufacturing process. Briefly, on day 0, tumor tissue is isolated from hypothermosol, the bioload sample is stored in transport medium, and tumor fragments are seeded into multiple (2, 3, or 4) G-Rex 100MCS culture flasks at a density ≤50 fragments/flask. Excess fragments are rapidly frozen. In some embodiments, the activation step can be incorporated into the pre-REP step, which provides a better co-stimulatory environment for TILs within the rosette/tumor ME. For example, on day 3, 60 μg of OKT3 or TransAct is added to each of the multiple G-Rex 100MCS culture flasks, and the cells undergo initial activation. On days 7/8, the volume is reduced, the sample is filtered, and transferred to a pooled EXP1000. The sample is removed for cell counting/viability analysis. Cells are washed, centrifuged at 400 g, 20 °C for 10 min, and aliquoted into TALEN and control samples at a ratio ≥9:1. Cells were resuspended in T buffer and electroporated at 10 × ^10⁶ cells/cup with TALEN mRNA (TALEN sample) or without RNA (control sample). Electroporation control and TALEN samples from each cup were seeded into G-Rex100M culture flasks containing 100 mL of medium (containing 6000 IU/mL IL-2) and incubated at 37°C for 1 hour. Feeder cells were irradiated, thawed, collected, and incubated with IL-2. Samples were removed for cell counting and viability analysis. Feeder cells and additional IL-2 and OKT3 were added to the incubated control and TALEN samples in G-Rex 100MCS culture flasks to generate REP cultures (1x G-Rex 100MCS for control REP culture, ≤9x G-Rex 100MCS for TALEN REP culture). On days 10/11, 500 mL of culture medium along with 3000 IU/mL IL-2 was added to each culture flask of the REP culture. Alternatively, the cell culture medium could be completely replaced with fresh medium. This step did not require transferring the cell suspension and did not use G-Rex 500MCS. On day 16, the volume was reduced, and the samples were collected. The samples were transferred via a blood filter, and the samples were removed for cell counting/viability analysis. Control samples were centrifuged, and the control final formulation was prepared and cryopreserved at a controlled rate. TALEN samples were processed using the LOVO system, and the TALEN residue final formulation was prepared and cryopreserved at a controlled rate. The prepared product samples were then subjected to quality control analysis.

This process has the following advantages: a) it eliminates the suspension transfer step for activation in the bag; b) it allows for 1/10 scale control; c) it eliminates in-process transfer to G-Rex10; d) it eliminates the overnight incubation step at 30°C, allowing for immediate reactivation (REP) of the medium at ambient temperature; and e) it eliminates one treatment day.

Example 22: A Phase 1/2 Study Evaluating the Efficacy and Safety of PD-1KO TIL Infusion

introduction

The phase 1/2 open-label study of PD-1 knockout tumor-infiltrating lymphocyte (PD-1KO TIL) products was conducted in participants with unresectable or metastatic melanoma or stage III or IV non-small cell lung cancer.

PD-1KO TIL product is an autologous TIL product that has undergone genome editing with transcription activator-like effector nucleases to disrupt the gene PDCD1 encoding PD-1. This protocol will evaluate PD-1KO TIL product as a treatment option for participants with unresectable or metastatic melanoma or stage III or IV NSCLC.

Research Principles

This study marks the first human investigation of the PD-1KO TIL product. The antitumor activity of the PD-1KO TIL product and its ability to directly target and kill tumor cells in a manner similar to non-genome-edited autologous TIL products were evaluated, with the potential for enhanced antitumor activity due to PDCD1 disruption.

Consistent with the hypothesis, enhanced antitumor activity of the PD-1KO TIL product was observed relative to non-genome-edited autologous TILs in a patient-derived xenograft mouse model. A proof-of-concept nonclinical study of the PD-1KO TIL product further demonstrated that TALEN-mediated PDCD1 mutagenesis was reproducible, correlated with reduced PD-1 cell surface expression, and indicated that the general biological characteristics of autologous TILs considered crucial for antitumor activity were either unaffected or positively affected by PD-1 gene knockout. In the absence of conventional nonclinical animal models associated with human TIL testing, in vitro studies supported the safety of the PD-1KO TIL product. Low off-target activity was detected at one of the top 20 potential off-target sites. Given the low mutation frequency and the fact that this site corresponds to a non-coding RNA not known to be associated with the disease, this is unlikely to pose a safety risk to the product. Additional in vitro studies showed no TALEN-related clonal chromosomal abnormalities following TALEN-mediated genome editing.

background

Unresectable or metastatic melanoma, as well as stage III and IV NSCLC, represent malignancies with a significant unmet medical need. Despite recent advances in melanoma treatment, including ICIs (e.g., anti-cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4] and anti-PD-1 monoclonal antibodies [mAb]) and targeted therapies (e.g., proto-oncogene B-Raf [BRAF] and mitogen-activated protein kinase [MEK] inhibitors), unresectable or metastatic melanoma remains difficult to treat and continues to be a major public health problem (Howlader 2020; Steininger 2021). While several effective NSCLC therapies are currently available, including ICIs (i.e., nivolumab and pembrolizumab), platinum-based double chemotherapy, and tyrosine kinase inhibitors (TKIs) or other targeted therapies against driver mutation-positive tumors, none have provided adequate activity and durable cancer control. Ultimately, most tumors progress despite the initial effectiveness of the available therapies. Therefore, despite the approval of ICI, there remains a significant unmet medical need for melanoma and NSCLC patients who have made progress on available standard care therapies.

Autologous TILs have demonstrated durable responses and have the potential to address a major unmet need in clinical trial participants with metastatic melanoma who have limited treatment options, including those with primary refractory to anti-PD-1/PD-L1 therapy (Rosenberg 2011; Larkin 2021; Sarnaik 2021). Similarly, preliminary data suggest that TILs elicit clinically meaningful responses in NSCLC participants who have experienced disease progression following a series of therapies, including ICIs, and in participants with epidermal growth factor receptor (EGFR) driver mutation-positive tumors (Creelan 2021; Schoenfeld 2021).

The antitumor activity of TILs can be inhibited through interactions between lymphocytes and the tumor microenvironment. Specifically, signaling that occurs when PD-1 on the surface of T cells binds to PD-L1 on tumor cells negatively impacts T cell antitumor activity. Consistent with this finding, various antibody therapies that specifically block the PD-1/PD-L1 pathway, such as the anti-PD-1 antibodies pembrolizumab and nivolumab, have shown potent activity in solid tumors, both as single agents and in combination with non-genome-edited autologous TILs. Notably, in addition to their promising anticancer activity, clinical studies of non-genome-edited autologous TIL therapy combined with anti-PD-1 antibodies in unresectable or metastatic melanoma, recurrent and/or metastatic head and neck squamous cell carcinoma, and persistent, recurrent, or metastatic cervical cancer provide preliminary evidence of the safety of PD-1 signaling inactivation in the context of adoptive cell therapy (ACT) with TILs, suggesting a safety profile consistent with that of individual therapies (O'Malley 2021). This is consistent with the results of other studies conducted to date in participants with metastatic melanoma (evaluating the combination of TIL therapy and immune checkpoint inhibition) (Besser 2013; Mullinax 2018) and a study investigating the administration of TIL therapy in combination with nivolumab in participants with NSCLC (Creelan 2021).

Systemic administration of anti-PD-1 antibodies generates adverse events (AEs) due to the non-specific upregulation of immune pathways via T cell activation and proliferation. As a potential alternative to this treatment, TALEN genome editing technology is used to specifically inactivate PDCD1 in autologous TILs, thereby producing PD-1KO TIL products, PD-1 gene knockout products. By limiting the inhibition of PD-1 signaling in therapeutic tumor-derived T cells, PD-1KO TIL product therapy is expected to provide a safer alternative to ICI therapy, which imparts systemic inhibition of the PD-1 pathway. Furthermore, compared to anti-PD-1 antibody regimens requiring repeated administration, PD-1KO TIL product therapy has the potential to offer clinical benefit as a one-time treatment.

Benefit/Risk Assessment

Currently, no clinical safety data are available for the PD-1KO TIL product, as this is the first human study of the PD-1KO TIL product. There are no clinical safety data from studies of ACT and autologous TILs with PD-1 gene knockout via genome editing; however, safety data are available from non-genome-edited TIL regimens administered alone or in combination with anti-PD-1 antibodies. Safety data from studies of non-genome-edited TIL products are considered relevant because they are manufactured using a derivative process of the PD-1KO TIL product manufacturing process, have the same formulation as the PD-1KO TIL product, and are administered as part of a regimen similar to the proposed PD-1KO TIL product regimen.

The risk and benefit assessment presented below is based on observations related to the administration of NMA-LD and IL-2, as reported in their respective prescribing information, and clinical experience with similar treatments to PD-1KO TIL products, specifically non-genome-edited autologous TIL products administered alone or in combination with anti-PD-1 antibodies, as part of a regimen similar to the proposed PD-1KO TIL product regimen.

Benefit assessment

Potential benefits to participants include the possibility of receiving a research intervention that could stop, slow, or reverse cancer progression in patient populations lacking effective treatment options.

Overall Benefits and Risks Conclusion

Considering the following, the benefit-risk profile of the PD-1KO TIL product treatment regimen is suitable for participants planned to be enrolled in the IOV-GM1-201 study:

●The known toxicities of non-genome-edited autologous TIL regimens (i.e., NMA-LD, IL-2, and non-genome-edited autologous TIL products) administered alone or in combination with anti-PD-1 antibodies are monitorable and manageable.

● Measures taken to minimize the risk to participants

● The lack of effective treatment options that provide durable benefits to the study population, and

●This new therapy has the potential to improve the antitumor activity and safety profile of existing TIL-based solid tumor treatment regimens and PD-1 inhibitor therapies.

Goals and End Points

Research Design

Overall Design

This is a phase 1/2, open-label, non-randomized, multi-cohort, multicenter study with a safety lead-in period, evaluating autologous TIL regimens using the PD-1KO TIL product in participants with unresectable or metastatic melanoma (cohort 1) or stage III or IV NSCLC (cohort 2). In each cohort, tumor samples were excised from each participant and cultured in vitro to produce IOV-4001. Participants were infused with the PD-1KO TIL product, followed by IL-2, after lymphocyte depletion chemotherapy including cyclophosphamide and fludarabine. During the safety lead-in period, the first three participants from either cohort will receive treatment in a staggered manner. Once each participant successfully completes the 28-day DLT observation period (starting at the time of PD-1KO TIL product infusion), the next participant may be authorized to continue with NMA-LD. Subsequent recruitment will follow a standard 3+3 downgrade design, allowing additional participants to join while evaluating the safety and tolerability data of the emerging PD-1KO TIL product. These safety data will be reviewed by the SRC, which comprises representatives of the sponsor and researchers from all recruited DLT-evaluable participants, to advise on dose level decisions. Overall safety will be monitored by an independent DSMB. Recruitment for both cohorts will continue after the first 3 to 6 participants have completed the DLT observation period and received DSMB approval; there will be no staggered periods between participants.

Scientific Principles of Research Design

The study was designed without a control group because these participants, who had undergone extensive pretreatment, had no alternative effective treatment options that could provide durable benefits to the study population. Although no comparisons were available, given the nature of the participants' advanced, refractory disease and the fact that the study intervention (autologous TIL regimen) was administered as a single, one-time therapy, any major tumor regression observed during the study could be presumed to be attributable to the study intervention.

The primary efficacy endpoint of the second phase of this study, namely the ORR according to RECIST v1.1, is the validated method for evaluating tumor response in a single-arm study.

Study population

Prospective approval of deviations from recruitment criteria is not permitted; this is also known as program waiver or exemption.

Key inclusion criteria

Participants are eligible to be included in the study only if they meet all of the following criteria:

1. Participants must be at least 18 years old when signing the informed consent form.

2. Participants who were histologically or pathologically diagnosed with unresectable or metastatic melanoma of stage IIIC, IIID, or IV (cohort 1) or stage III or IV NSCLC (cohort 2).

3. Participants who have previously received the following treatments:

Cohort 1 (Melanoma): Participants who experienced documented radiographic disease progression during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibody. If the tumor was BRAF V600 mutation-positive, participants also received a BRAF inhibitor with or without a MEK inhibitor.

Cohort 2 (NSCLC): Participants who had previously received no more than 3 lines of prior therapy, and

● Participants without oncogene-driven tumors who experienced documented progression of radiation-related disease during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibodies.

● Or for participants with tumors driven by oncogenes for which effective targeted therapies are available (e.g., EGFR, anaplastic lymphoma kinase [ALK], ROS proto-oncogene [ROS], or proto-oncogenes encoding MET tyrosine receptor kinase [MET]), who have experienced documented radiological disease progression during or after at least one line of appropriate targeted therapy and platinum-based dual chemotherapy, or during systemic therapy with PD-1/PD-L1 blocking antibodies, or within 12 weeks after the last dose of a PD-1/PD-L1 blocking antibody.

4. Participants whose ECOG fitness level is 0 or 1.

5. Participants assessed as having at least one resectable lesion

6. Previously irradiated lesions must have radiographic progression before collection.

7. Participants who are of childbearing age or whose partners are of childbearing age must be willing to use an approved, highly effective method of contraception during the treatment period and for 12 months after receiving all protocol-related treatments.

Key Exclusion Criteria

Participants were excluded from the study if they met any of the following criteria:

1. Participants with severe heart disease.

2. Participants with uveal/ocular melanoma.

3. Participants with symptomatic and untreated brain metastases.

4. Participants with any form of primary immunodeficiency.

5. Participants who have had another primary malignant disease within the previous 3 years.

6. Participants who have received or will receive a live or attenuated vaccine within 28 days prior to the start of NMA-LD.

References for Example 22

Besser MJ, Shapira-Frommer R, ltzhaki O, et al. Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma: Intent-to-Treat Analysis and Efficacy after Failure to Prior Immunotherapies. ClinCancer Res. 2013; 19(17): 4792-800.

Creelan BC, Wang C, Teer J, et al. Tumor-Infiltrating LymphocyteTreatment for Anti-PD-1-Resistant Metastatic Lung Cancer: A Phase 1 Trial. NatMed. 2021; 27(8): 1410-1418.

Howlader N, Noone A, Krapcho M, et al. 2020. SEER Cancer Statistics Review, 1975-2017. National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2017/, based on November 2019 SEER data submission.

Larkin JMG, Sarnaik A, Chesney JA, et al. Lifileucel(LN-144), aCryopreserved Autologous Tumor Infiltrating Lymphocyte(TIL)Ther apy inPatients with Advanced Melanoma: Evaluation of Impact of Prior Anti-PD-1Therapy.J Clin Oncol.2021;39(15_suppl):9505.

Mullinax JE, Hall M, Prabhakaran S, et al. Combination of lpilimumab and Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes for Patients withMetastatic Melanoma. Front Oncol. 2018; 8:44.

O'Malley D, Lee SM, Psyrri A, et al.Phase 2Efficacy and Safety ofAutologous Tumor-Infiltrating Lymphocyte(TIL)Cell Therapy in Combinati on with Pembrolizumab in Immune Checkpoint Inhibitor-Patients with Advanced Cancers J Immunother Cancer. 2021; 9(suppl 2, abs492): A523-4.

Rosenberg SA, Yang JC, Sherry RM, et al. Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell TransferImmunotherapy. Clin Cancer Res. 2011;17(13):4550-7.

Sarnaik AA, Hamid O, Khushalani NI, et al. Lifileucel, a Tumor-Infiltrating Lymphocyte Therapy, in Metastatic Melanoma. J Clin Oncol. 2021: JCO2100612.

Schoenfeld AJ, Lee SM, Paz-Ares L, et al. First Phase 2Results of Autologous Tumor-Infiltrating Lymphocyte (TIL; LN-145) Monotherapy in Patient swith Advanced, Immune Checkpoint Inhibitor-Treated, Non-Small Cell Lung Cancer (NsCLC). J Immunother Cancer. 2021; 9 (suppl2, abs 458): A486-7.

Steininger J, Gellrich FF, Schulz A, et al. Systemic Therapy of Metastatic Melanoma: On the Road to 75Cure. Cancers (Basel). 2021; 13(6).

Example 23: Evaluation of Xenon Electroporation Instrument

A study was conducted to determine whether a thermoexcimer could be used in the process of generating PD-1KO TILs. The results are summarized in Figure 76.

Method Overview

Activated or pre-REP TILs are used for experiments.

●REP TIL: Demonstration Day, Neon Experiment 1, Xenon Experiment 1

●Pre-REP TIL: Xenon Experiment 3, Xenon Experiment 4

●Activate for 5 days using TransAct

Cell preparation days for electroporation (same across experiments):

● Equilibrate the buffer solution to room temperature.

● Wash TIL with 1x PBS

● Wash TIL with 1x electroporation buffer (Thermo-CTS ^™ Xenon ^™ Genome Editing Buffer)

● Centrifuge and resuspend in the final volume of electroporation buffer.

○ A single injection requires an exact volume of 1 ml per sample (maximum 100e6/ml).

○ The neon volume can be 10ul or 100ul (maximum 20e6/ml).

●Add mRNA (GFP 1ug/1e6 cells, TALEN 4ug/1e6 cells)

●Electroplution

● Rest overnight in CM2 + 3000 IU/ml IL-2 in G-Rex10 (if using >10e6, split into 2 G-Rex10s).

●37℃, for GFP mRNA

●30℃, for TALEN mRNA

● Collect resting samples and bacterial strains using a 150µm cell filter.

● Perform cell counting and flow staining or REP (Xenon Experiment 4).

Experiment 1: Demonstration Day, Thermo

● Activated REP TIL (L4346) was tested using GFP mRNA.

● Thawing (Day 0) and Activation (Day 1) 50e6+TransAct

● 1ug/1e GFP mRNA per 6 cells (stock solution, 1mg/ml)

● Electroporation using Thermo's recommended optimization scheme

● Single injection (range: 2e7-1e8 cells per 1ml) – 20e6/ml

●Neon – 2e6/100ul

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 77, the results indicate that after 24 hours, 1700/20/1 and 2300/3/4 showed higher GFP expression and recovery rates.

Experiment 2: Neon Experiment 1

●Objective: To optimize two electroporation setups identified during the xenon demonstration using neon and compare them with BTX.

●Cell concentration: 2e6 cells per 100 μl of neon-1

● Xenon backbone electroporation setup:

○1700/20/1

○2300/3/4

● Change the duration (ms) and number of pulses while keeping the voltage setting constant.

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 78, the results indicate that the 2300/3/4 Thermo recommended setting (5*) has the highest GFP expression and 60% recovery rate, and the recovery rate is improved at the 2300V voltage setting with shorter duration (2ms) and fewer pulses (3), and GFP expression is reduced.

Experiment 3: Xenon Experiment 1

●Objective: To determine the effect of cell concentration on xenon-GFP expression and recovery rate.

●N = 1 activated REP TIL sample (EP11231)

●Xenon and BTX

●Electroporation conditions: 2300/3/4, selected based on maximum GFP expression and 60% recovery rate.

●Use a single injection to test cell concentration

○1e6/ml

○5e6/ml

○10e6/ml

○25e6/ml

●BTX Comparison

○10e6+GFP (in 400 μl, therefore the concentration is 25e6/ml)

○2e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 79, the results indicate that GFP expression was slightly reduced when xenon was used compared to BTX. GFP expression was not affected by cell concentration, but the recovery rate decreased and apoptosis increased at lower cell concentrations. After 24 hours, 2300/3/4 appeared to be toxic at low cell concentrations.

Experiment 4: Xenon Experiment 3

Objective: To determine the effect of cell concentration (low/high) on xenon GFP expression and recovery rate using the EP setting that exhibits the highest recovery rate.

●N = 1 activated pre-REP TIL sample (K7136)

●Xenon and BTX

●Electroplution conditions:

○1400/30/1

○1700/20/1

○2300/2/3

○2500/2/5

●Use a single injection to test cell concentration

○5e6/ml

○25e6/ml

●BTX Comparison

○5e6+GFP (in 200 μl, therefore the concentration is 25e6/ml)

○1e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 80, the results indicate that high GFP expression exists between samples, comparable to that of BTX, and the difference between 5e6 and 25e6 is minimal. For EP settings 1400/30/1 and 2300/2/3, there are similar recovery rates between 5e6 and 25e6, and after 24 hours, the recovery rate of 2300/2/3 is comparable to that of BTX.

Experiment 5: Xenon Experiment 4

●Objective: To compare the Xenon electroporator and the BTX electroporator using TALEN mRNA.

●N = 1 activated pre-REP TIL sample (L4374)

●Xenon and BTX

●Electroporation conditions:

○2300/2/3

●Conditions:

Xenon (Talen(25e6))

Xenon simulant (5e6)

○BTX Talen (25e6)

○BTX emulator (5e6)

●Test parameters:

○Resuscitation rate and survival rate

○ PD-1 KO based on flow cytometry

■ PD-1 phase KO flow cytometry data operated on resting samples after electroporation (without REP)

■REP collects final PD-1KO flow cytometry data from samples

As shown in Figure 81, the results showed that the recovery rates of xenon and BTX were similar between 5e6 and 25e6, with xenon having a slightly higher recovery rate. The PD-1 KO efficiency (resting cells after electroporation) was similar between xenon (89%) and BTX (86%). Furthermore, for the analysis of PD-1 expression and KO efficiency in REP samples, the KO efficiency of xenon (83%) was slightly higher than that of BTX (76%).

in conclusion

The study identified an electroporation setting of 2300/2/3 for xenon that resulted in high cell resuscitation rates (comparable to BTX) at both low and high cell concentrations, high GFP expression (comparable to BTX) at both low and high cell concentrations, and high PD-1 KO efficiency (comparable to BTX).

The above embodiments are provided to give those skilled in the art a complete disclosure and description of how to prepare and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications to the above-described patterns of the invention that are obvious to those skilled in the art are intended to fall within the scope of the following claims. All patents and disclosures mentioned in this specification indicate the skill level of those skilled in the art to which this invention pertains.

All headings and section titles are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different headings and sections as needed, in accordance with the spirit and scope of the invention described herein.

All references cited in this article are incorporated herein by reference in their entirety for all purposes, as if each individual publication or patent or patent application were specifically and individually indicated to be incorporated herein by reference in their entirety for all purposes.

As will be apparent to those skilled in the art, various modifications and alterations can be made thereto without departing from the spirit and scope of this application. The specific embodiments and examples described herein are provided by way of example only, and this application is limited only to the appended claims and the full scope of their equivalents.

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 63/242,373, filed September 9, 2021; U.S. Provisional Application No. 63/287,670, filed September 9, 2021; U.S. Provisional Application No. 63/322,190, filed March 21, 2022; U.S. Provisional Application No. 63/354,605, filed June 22, 2022; and U.S. Provisional Application No. 63/394,248, filed August 1, 2022, the disclosures of which are incorporated herein by reference in their entirety.

Background of the Invention

Using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) to treat large, refractory cancers represents an effective approach for treating patients with poor prognoses. (Gattinoni et al., Nat. Rev. Immunol. 2006, 6, 383-393). TILs are predominantly T-cells, and IL-2-based TIL expansion followed by a “rapid expansion process” (REP) is the preferred method for TIL expansion due to its speed and efficiency. (Dudley et al., Science 2002, 298, 850-54; Dudley et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. Immunother. 2003, 26, 332-42.) Numerous approaches have been explored to improve the response of melanoma to TIL therapy and to extend TIL therapy to other tumor types, but success has been limited and the field remains challenging. (Goff et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg et al., Clin. Cancer Res. 2011, 17, 4550-57.) Combination studies with single immune checkpoint inhibitors have also been described, but further investigation is underway and additional treatment options are needed (Kverneland et al., Oncotarget, 2020, 11(22), 2092-2105).

Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, aseptic issues, and other factors described herein, severely restricting their therapeutic potential for patients refractory to checkpoint inhibitor therapy. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes, suitable for treating patients with few or no viable treatment options. This invention addresses this need by providing a shortened manufacturing process for generating TILs.

The present invention provides improved and/or shortened processes and methods for amplifying TILs and generating therapeutic TIL populations, including methods for gene editing at least a portion of the therapeutic TIL population to enhance its therapeutic efficacy.

Summary of the Invention

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing of at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, the culturing or rapid second expansion of the fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the fourth TIL population into multiple subcultures; each subculture being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs or a therapeutic TIL population.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the activation of the second TIL population is performed using anti-CD3 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using OKT-3.

In some implementations, the step of activating the second TIL population is performed using 300 ng/mL of OKT-3.

In some implementations, the step of activating the second TIL population is performed using anti-CD3 agonist and anti-CD28 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using TransAct.

In some implementations, the step of activating the second TIL population is performed using TransAct diluted at 1:10, 1:17.5, or 1:100.

In some implementations, the step of activating the second TIL population takes about 2 days.

In some implementations, the step of activating the second TIL population takes about 3 days.

In some implementations, the step of activating the second TIL population takes approximately 4 days.

In some implementations, the step of activating the second TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the fourth TIL population takes about 8 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 9 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the step of culturing the fourth TIL population takes about 10 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A second TIL population was produced by culturing a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium for about 3–9 days in a first cell culture medium containing IL-2 and OKT-3;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some implementations, the step of culturing or rapidly expanding the third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the third TIL population into multiple subcultures; each of the subcultures being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the third TIL population takes about 8 days.

In some implementations, the step of culturing the third TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes about 8-9 days.

In some implementations, the step of culturing the third TIL population takes about 10 days.

In some implementations, the step of culturing the third TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, the step of culturing or initially amplifying the first TIL population in a first culture medium also includes anti-CD3 and anti-CD28 beads or antibodies.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population for about 3 days in a first cell culture medium containing IL-2 and anti-CD3 and anti-CD28 beads or antibodies, followed by culturing the first TIL population for 2-4 days in a cell culture medium containing IL-2 and OKT-3.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of at least two gene editors is transferred independently of the transfer of any other gene editor during the electroporation step.

In some implementations, the electroporation step also includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing IL-2 and/or IL-15.

In some embodiments, the first and second resting periods comprise incubating a third or fourth TIL population in a second cell culture medium containing 300 IU/mL, 1000 IU/mL, or 6000 IU/mL of IL-2.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing 15 ng/mL IL-15.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first and second resting periods comprise incubating a third or fourth TIL population at approximately 25, 28, 30, 32, 35, or 37°C with approximately 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some implementations, the electroporation step is preceded by washing a second or third TIL population in a cell pore buffer.

In some embodiments, the at least one gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates PD-1 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CTLA-4 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates LAG-3 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CISH expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CBL-B expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CTLA-4.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and TIGIT.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CISH and CBL-B.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, the tumor tissue is processed into multiple tumor fragments.

In some implementations, the tumor fragment is added to the closed system.

In some embodiments, 150 or fewer fragments, 100 or fewer fragments, or 50 or fewer fragments are added to the closed system. 167. A gene-edited tumor-infiltrating lymphocyte (TIL) population comprising an expanded TIL population, wherein the expression of at least one protein is regulated by a gene editor transferred to at least a portion of the expanded TIL population.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one protein is PD-1.

In some embodiments, the at least one protein is CTLA-4.

In some embodiments, the at least one protein is LAG-3.

In some embodiments, the at least one protein is CISH.

In some embodiments, the at least one protein is CBL-B.

In some embodiments, the at least one protein is TIGIT.

In some embodiments, the expression of the at least two proteins is regulated by at least two gene editors transferred to at least a portion of the amplified TIL population, wherein the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of the first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of the second protein.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first TALE protein and the second TALE protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some implementations, the gene-edited TIL populations disclosed herein are created using the methods disclosed herein.

In some embodiments, this document provides a pharmaceutical composition comprising the gene-edited TIL population disclosed herein and a pharmaceutically acceptable vector.

In some implementations, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of the gene-edited TIL population disclosed herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) The tumor fragment is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step includes delivering a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of the TALEN system for inhibiting CTLA-4 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CBL-B and TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is about 1× ^10⁹ to about 1× ^10¹¹ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of the collected TIL population in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the patient has been given a therapeutically effective dose of the collected TIL population in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Attached Figure Description

Figure 1: Exemplary Gen 2 (Process 2A) diagram, which provides an overview of steps A through F.

Figures 2A to 2C: Process flow diagrams of one implementation of Gen 2 (process 2A) for TIL manufacturing.

Figure 3: A diagram showing one embodiment of an exemplary manufacturing process for cryopreserved TILs (approximately 22 days).

Figure 4: A diagram showing one implementation of Gen 2 (Process 2A, a 22-day process for TIL manufacturing).

Figure 5: Comparison table of steps A to F of an exemplary implementation of process 1C and Gen 2 (process 2A) for TIL manufacturing.

Figure 6: A detailed comparison of one implementation of Process 1C and one implementation of Gen 2 (Process 2A) for TIL manufacturing.

Figure 7: Exemplary Gen 3 type TIL manufacturing process.

Figures 8A through 8P: A) A comparison between an implementation of the 2A process (approximately 22 days) and a Gen 3 process (approximately 14 to 16 days) for TIL manufacturing. B) An exemplary Gen 3 process diagram providing an overview of steps A through F (approximately 14 to 16 days). C) Diagrams of three exemplary Gen 3 processes, outlining steps A through F (approximately 14 to 16 days) for each of the three process variations. D) An exemplary modified Gen 2-like process providing an overview of steps A through F (approximately 22 days). E-P) Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 9: Experimental flowchart providing comparability between Gen 2 (process 2A) and Gen 3 processes.

Figure 10: A comparison of various Gen 2 (Process 2A) and Gen 3.1 process implementation schemes.

Figure 11: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 12: An overview of the culture medium conditions for one implementation of the Gen 3 process (referred to as Gen 3.1).

Figure 13: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 14: A table comparing various features of the implementation schemes for the Gen 2 and Gen 3.0 processes.

Figure 15: A table showing the culture medium used in various implementation schemes of the described amplification process.

Figure 16: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 17: A schematic diagram of an exemplary implementation of a method for amplifying T cells from hematopoietic malignancies using the Gen 3 amplification platform.

Figure 18: Structures IA and IB are provided. The cylinder refers to an individual polypeptide-binding domain. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from, for example, 4-1BBL or antibodies binding to 4-1BB, which fold to form a trivalent protein. This trivalent protein is then linked to a second trivalent protein via an IgG1-Fc (including CH3 and CH2 domains), which in turn links the two trivalent proteins together via disulfide bonds (small elongated ellipses). This stabilizes the structure and provides an agonist capable of aggregating the intracellular signaling domains of six receptors with signaling proteins to form a signaling complex. The TNFRSF-binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys sequences.

Figure 19: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 20: Process overview of an exemplary implementation of the Gen 3.1 process (16-day process).

Figure 21: A schematic diagram of an exemplary implementation of the Gen 3.1 testing process (16-day to 17-day process).

Figure 22: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 23: Comparison table of exemplary Gen 2 and exemplary Gen 3 processes.

Figure 24: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day to 17-day process) preparation timeline.

Figure 25: A schematic diagram of an exemplary implementation of the Gen 3 process (14-day to 16-day process).

Figures 26A to 26B: Schematic diagrams of an exemplary implementation of the Gen 3 process (16-day process).

Figure 27: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 28: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 29: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 30: Components of the Gen 3 implementation scheme.

Figure 31: Comparison of Gen 3 implementation flowcharts (Gen 3.0, Gen 3.1 comparison, Gen 3.1 test).

Figure 32: Shows components of an exemplary implementation of the Gen 3 process (16- to 17-day process).

Figure 33: Acceptance Criteria Table.

Figure 34: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figure 35: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figures 36A to 36D: Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 37: A schematic diagram of an exemplary implementation of the process described in Example 12.

Figures 38A-38B: In vivo efficacy of PDCD-1KO TIL. A) PDCD-1KO efficiency as assessed by flow cytometry. B) hIL-2NOG mice with melanoma tumor cells transplanted via adoptive transfer using PDCD-1KO or TIL-mimicking mice (n=14 per treatment group). Anti-PD-1 antibody treatment combined with TIL-mimicking mice served as a control for PD-1/PD-L1 blockade. Statistical significance is indicated by *p<0.05, **p<0.01, and ****p<0.0001.

Figures 39A to 39E: TIL product analysis. A) Live cell dose, B) Purity, C) Identity, D) Potency, and E) PDCD-1KO efficiency of TIL products.

Figures 40A to 40B: TIL product analysis. A) TIL differentiation and B) TIL memory.

Figures 41A to 41B: Expression of activation and inhibition of related biomarkers on PDCD-1KO TIL.

Figures 42A to 42B: IL-2-independent proliferation assay of PDCD-1KO TIL products.

Figure 43: Summary of karyotype analysis results of PDCD-1KO TIL products.

Figures 44A to 44B: Cell viability (Figure 44A) and recovery fold (Figure 44B) before electroporation.

Figures 45A to 45B: Cell recovery fold (Figure 45A) and cell viability (Figure 45B) after electroporation.

Figures 46A to 46C: Gene knockout efficiency of CD3+ (Figure 46A), CD8+ (Figure 46B), and CD4+ (Figure 46C) cells.

Figures 47A to 47B: Cell recovery fold (Figure 47A) and cell viability (Figure 47B) after electroporation.

Figures 48A to 48B: Cell recovery fold (Figure 48A) and cell viability (Figure 48B) after electroporation when using 6000 IU/mL IL-2.

Figures 49A to 49B: Cell recovery fold (Figure 49A) and cell viability (Figure 49B) after electroporation under various conditions.

Figures 50A to 50C: Gene knockout efficiency of CD3+ (Figure 50A), CD8+ (Figure 50B), and CD4+ (Figure 50C) cells.

Figure 51: Cell viability before electroporation.

Figure 52: Cell resuscitation fold before electroporation.

Figures 53A to 53B: Cell recovery fold (Figure 53A) and cell viability (Figure 53B) after electroporation.

Figures 54A to 54C: Gene knockout efficiency of CD3+ (Figure 54A), CD8+ (Figure 54B), and CD4+ (Figure 54C) cells.

Figures 55A to 55B: Cell number (Figure 55A) and survival rate (Figure 55B) after various washing steps.

Figures 56A to 56B: Cell numbers after various rotation conditions using PBS washing (Figure 56A) or Cyto washing (Figure 56B).

Figures 57A to 57B: Cell viability after various rotation conditions using PBS washing (Figure 57A) or Cyto washing (Figure 57B).

Figures 58A to 58B: Total rotation comparison of cell number (Figure 58A) and total rotation comparison of cell survival rate (Figure 58B) after various rotation conditions.

Figure 59: Percentage of cell loss after total rotation under various rotation conditions.

Figures 60A to 60C: Percentage of cell loss and survival rate during electroporation, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation.

Figures 61A to 61C: Gene knockout efficiency of CD3+ (Figure 61A), CD8+ (Figure 61B), and CD4+ (Figure 61C) cells.

Figures 62A to 62B: Cell viability (Figure 62A) and fold expansion (Figure 62B) of the REP collection.

Figures 63A to 63B: Percentage of cell loss after electroporation (Figure 63A) and cell viability (Figure 63B).

Figures 64A to 64C: Gene knockout efficiency of CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells.

Figures 65A to 65B: Folding of REP collection (Figure 65A) and cell viability (Figure 65B).

Figures 66A to 66C: Cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C).

Figure 67: Percentage of growth after 3 days of rest.

Figures 68A to 68C: PD-1 gene knockout efficiency.

Figure 69: PDCD1 gene modification according to NGS.

Figures 70A to 70B: Distribution of the TCR Vβ isotype in the CD3+PD-1- subset in the main PD-1KO TIL product and NE TIL.

Figures 71A to 71B: PD-1KO TIL effect function as measured by MLR (Figure 71A) and multifunctionality (Figure 71B).

Figure 72: In vivo antitumor activity of M1152 PD-1KO TIL product.

Figures 73A to 73B: Durability of TALEN protein in autologous TILs as a function of time, measured by Western blot.

Figures 74A-F: Exemplary TIL manufacturing process.

Figures 75A-B: Schematic diagrams of the 1/2 phase study described in Example 22.

Figure 76: Summary of data described in Example 23.

Figures 77A-D: Results of the demonstration day experiment from Example 23.

Figures 78A-C: Results of neon experiment 1 from Example 23.

Figures 79A-C: Results of Xenon Experiment 1 from Example 23.

Figures 80A-B: Results of Xenon Experiment 3 from Example 23.

Figures 81A-C: Results of Xenon Experiment 4 from Example 23.

A brief explanation of sequence listings

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of moromumab.

SEQ ID NO:3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO:4 is the amino acid sequence of adefovir.

SEQ ID NO:5 is in IL-2 form.

SEQ ID NO:6 is the amino acid sequence of Nevo interleukin-α.

SEQ ID NO:7 is in IL-2 form.

SEQ ID NO:8 is a mucin domain polypeptide.

SEQ ID NO:9 is the amino acid sequence of recombinant human IL-4 protein.

SEQ ID NO:10 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO:11 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO:12 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO:13 is an IL-2 sequence.

SEQ ID NO:14 is the sequence of the IL-2 mutant protein.

SEQ ID NO:15 is the sequence of the IL-2 mutant protein.

SEQ ID NO:16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

SEQ ID NO:17 is HCDR2 of IgG.IL2R67A.H1.

SEQ ID NO:18 is HCDR3 of IgG.IL2R67A.H1.

SEQ ID NO:19 is HCDR1_IL-2kabat of IgG.IL2R67A.H1.

SEQ ID NO:20 is HCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:21 is HCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:22 is HCDR1_IL-2clothia of IgG.IL2R67A.H1.

SEQ ID NO:23 is HCDR2 clothia of IgG.IL2R67A.H1.

SEQ ID NO:24 is HCDR3 clothia of IgG.IL2R67A.H1.

SEQ ID NO:25 is HCDR1_IL-2IMGT of IgG.IL2R67A.H1.

SEQ ID NO:26 is HCDR2 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:27 is HCDR3 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:28 is the V _H chain of IgG.IL2R67A.H1.

SEQ ID NO:29 is the heavy chain of IgG.IL2R67A.H1.

SEQ ID NO:30 is the LCDR1 kabat of IgG.IL2R67A.H1.

SEQ ID NO:31 is the LCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:32 is the LCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:33 is the LCDR1 chothia of IgG.IL2R67A.H1.

SEQ ID NO:34 is the LCDR2 chothia of IgG.IL2R67A.H1.

SEQ ID NO:35 is the LCDR3 chothia of IgG.IL2R67A.H1.

SEQ ID NO:36 is the V _L chain.

SEQ ID NO:37 is a light chain.

SEQ ID NO:38 is a light chain.

SEQ ID NO:39 is a light chain.

SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

SEQ ID NO:41 is the amino acid sequence of mouse 4-1BB.

SEQ ID NO:42 is the heavy chain of utomilumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:43 is the light chain of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:44 is the heavy chain variable region (V _H ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:45 is the light chain variable region (V _L ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:46 is the heavy chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:47 is the heavy chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:48 is the heavy chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:49 is the light chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:50 is the light chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:51 is the light chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:52 is the heavy chain of urelumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:53 is the light chain of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:54 is the heavy chain variable region (VH) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:55 is the light chain variable region (VL) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:56 is the heavy chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:57 is the heavy chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:58 is the heavy chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:59 is the light chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:60 is the light chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:61 is the light chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:62 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:63 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:64 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:65 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:66 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:67 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:68 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:69 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:70 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:71 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:72 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:73 represents the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:74 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:75 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:76 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:77 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

SEQ ID NO:78 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO:79 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 1.

SEQ ID NO:80 is the light chain variable region (V _L ) of type 1 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:81 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 2.

SEQ ID NO:82 is the light chain variable region (V _L ) of type 2 of 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:83 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:84 is the light chain variable region (V _L ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:85 is the amino acid sequence of human OX40.

SEQ ID NO:86 is the amino acid sequence of mouse OX40.

SEQ ID NO:87 is the heavy chain of the OX40 agonist monoclonal antibody taboliziumab (MEDI-0562).

SEQ ID NO:88 is the light chain of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:89 is the heavy chain variable region (V _H ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:90 is the light chain variable region (V _L ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:91 is the heavy chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:92 is the heavy chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:93 is the heavy chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:94 is the light chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:95 is the light chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:96 is the light chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:99 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:100 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:107 is the heavy chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:108 is the light chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:109 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:110 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:117 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:118 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:119 is the heavy chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:120 is the heavy chain CDR2 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:122 is the light chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:125 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:126 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:130 is the light chain CDR1 of Hu106-222, an OX40 agonist monoclonal antibody.

SEQ ID NO:131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:133 is the amino acid sequence of the OX40 ligand (OX40L).

SEQ ID NO:134 is the soluble portion of the OX40L polypeptide.

SEQ ID NO:135 is an alternative soluble fraction of the OX40L polypeptide.

SEQ ID NO:136 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:137 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:138 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:139 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:140 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 021.

SEQ ID NO:141 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 021.

SEQ ID NO:142 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:143 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:144 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:145 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:146 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:147 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:148 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:149 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:150 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:151 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:152 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:153 is the heavy chain variable region ( _VH ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:154 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:155 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:156 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:157 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:160 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:161 is the amino acid sequence of the light chain variable region ( _VL ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:170 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:171 is the amino acid sequence of the light chain variable region (V _L ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:180 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:181 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:190 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:191 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:200 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:201 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:210 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:211 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:220 is the amino acid sequence of the heavy chain variable region (V _H ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:221 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4 inhibitor zeflimab.

SEQ ID NO:230 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:231 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:238 is an exemplary Clo05 l nuclease domain amino acid sequence.

SEQ ID NO:239 is an exemplary piggyBac(PB) transposase amino acid sequence.

SEQ ID NO:240 is an exemplary Sleeping Beauty transposase amino acid sequence.

SEQ ID NO:241 is an exemplary amino acid sequence of an overactive Sleeping Beauty (SB100X) transposase.

I. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and disclosures mentioned herein are incorporated herein by reference in their entirety.

As used herein, the terms "co-administration/co-administering," "administered in combination with/administering in combination with," "simultaneous," and "concurrent" encompass the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, such as multiple TILs) to a subject, such that both the active pharmaceutical ingredient and/or its metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of separate compositions and administration of a composition in which two agents are present are preferred.

The term "in vivo" refers to events that occur within the body of a subject.

The term "in vitro" refers to an event that occurs outside the subject's body. In vitro assays encompass cell-based assays using live or dead cells, and may also include cell-free assays that do not use intact cells.

The term "ex vivo" refers to an event involving the treatment or procedure performed on cells, tissues, and/or organs that have been removed from a subject's body. Appropriately, cells, tissues, and/or organs may be returned to the subject using surgical or therapeutic methods.

The term "rapid amplification" means that the number of antigen-specific TILs increases at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) within one week, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) within one week, or most preferably at least about 100-fold within one week. Several rapid amplification protocols are described herein.

In this article, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are TILs obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any expanded or proliferated population of TIL cells as discussed herein, including (but not limited to) main TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations may contain genetically modified TILs.

In this article, "cell population" (including TILs) refers to a large number of cells sharing common characteristics. Typically, the population size ranges from 1 × ^10⁶ to 1 × ^10¹⁰ cells, with different TIL populations containing varying numbers. For example, initial growth of primary TILs in the presence of IL-2 produces a main TIL population of approximately 1 × ^10⁸ cells. REP expansion is typically performed to provide a cell population of 1.5 × ^10⁹ to 1.5 × ^10¹⁰ cells for infusion.

In this document, “cryopreserved TIL” means TIL that has been processed and stored in the range of approximately -150°C to -60°C, whether primary, host, or amplified (REP TIL). General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, “cryopreserved TIL” may be distinguished from frozen tissue samples that can be used as a source of primary TIL.

In this article, “thawed cryopreserved TILs” means a population of TILs that were previously cryopreserved and then processed to be restored to room temperature or higher (including, but not limited to, cell culture temperatures or temperatures at which TILs can be administered to patients).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to reintroduce into patients with late-stage invasive solid tumors.

The term "cryopreservation media" refers to any culture medium that can be used for cryopreservation of cells. Such media may include those containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 media may be referred to by the trade name "CS10". CS10 media is a serum-free, animal-free medium containing DMSO. In some embodiments, CS10 media contains 10% DMSO.

The term "central memory T cells" refers to a subset of T cells in humans that are CD45R0+ and constitutively express CCR7 (CCR7 ^hi ) and CD62L (CD62 ^hi ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Upon TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are mainly found in the CD4 compartments of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

The term "effective memory T cells" refers to a subset of human or mammalian T cells, such as central memory T cells, which are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 ^lo ) and are heterogeneous or low in expression of CD62L (CD62L ^lo ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, upon antigen stimulation. Effector memory T cells are primarily found in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry a large amount of perforin.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, for example (but not limited to), closed G containers. Once a tumor segment is added to the closed system, the system remains closed to the external environment until the TIL is ready for administration to the patient.

As used herein, the terms “fragmenting,” “fragment,” and “fragmented” describe the process of destroying a tumor, including mechanical fragmentation methods such as crushing, slicing, splitting, and pulverizing tumor tissue, as well as any other methods used to destroy the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBL is separated from whole blood or apheresis products from a donor. In some embodiments, PBL is separated from whole blood or apheresis products from a donor by positively or negatively selecting for a T cell phenotype (e.g., CD3+CD45+ T cell phenotype).

The term "anti-CD3 antibody" refers to an antibody or a variant thereof that targets the CD3 receptor in the T-cell antigen receptor of mature T cells, such as a monoclonal antibody, and includes human, humanized, chimeric, murine, or mammalian antibodies. Anti-CD3 antibodies include OKT-3, also known as moromuzumab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof targeting the CD3 receptor in the T-cell antigen receptor of mature T cells, including human, humanized, chimeric, or murine antibodies, and including commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, San Diego, CA, USA) and moromumab or its variants, conserved amino acid substitutions, glycosylated forms, or biosimilars. The amino acid sequences of the heavy and light chains of moromumab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). Hybridomas capable of producing OKT-3 are deposited at the American Type Culture Collection and designated ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited at the European Collection of Authenticated Cell Cultures (ECACC) with catalog number 86022706.

Table 1. Amino acid sequence of moromuzumab (exemplary OKT-3 antibody)

The term "IL-2" (also referred to herein as "IL2") refers to a T-cell growth factor called interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in this invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses recombinant human IL-2, such as adefovir (PROLEUKIN, available from multiple suppliers, 22 million IU per single-use vial), as well as recombinant IL-2 forms supplied by CellGenix (CELLGRO GMP) of Portsmouth, NH, USA, or ProSpec-Tany TechnoGene Ltd. (catalog number CYT-209-b) of East Brunswick, NJ, USA, and other commercial equivalents from other suppliers. Adefovir (desalanyl-1, serine-125 human IL-2) is a non-glycosylated recombinant human IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of the adefovir suitable for use in this invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including the PEGylated IL-2 prodrug beempegaldesleukin (NKTR-214, as SEQ ID NO:4, PEGylated recombinant human IL-2 wherein an average of 6 lysine residues are substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluorene-9-yl)methoxy]carbonyl N ⁶ ), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO 2018/132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US2019/0275133 A1, the disclosure of which is incorporated herein by reference. The bepedelion interleukin (NKTR-214) and other PEGylated IL-2 molecules applicable to the present invention are described in U.S. Patent Application Publication No. US2014/0328791A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 applicable to the present invention are described in U.S. Patents Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations applicable to the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

In some embodiments, the IL-2 form suitable for use in this invention is THOR-707, available from Synthorx. The preparation and characteristics of THOR-707 and additional alternative forms of IL-2 suitable for use in this invention are described in U.S. Patent Application Publications Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for use in this invention is an interleukin-2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugate portion bound to the isolated and purified IL-2 polypeptide at amino acid positions selected from the following: K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the amino acid residues are numbered corresponding to SEQ ID NO: 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residues are mutated to cysteine. In some embodiments, the amino acid residues are mutated to lysine. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to non-natural amino acids. In some embodiments, the non-natural amino acids include N6-azidoethoxy-L-lysine (AzK), N6-propynylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, 3-methyl-phenylalanine, L-DOPA, fluorinated phenylalanine, Isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzoxy)-3-oxopropyl)amino)ethyl)selenoyl)propionic acid, 2-amino-3-(phenylselenoyl)propionic acid, or selenocysteine. In some embodiments, the IL-2 conjugate exhibits reduced affinity for the IL-2 receptor α (IL-2Rα) subunit relative to the wild-type IL-2 peptide. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% of the binding affinity to IL-2Rα. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 100, 200, 300, 500, 1000, or greater. In some embodiments, the conjugated moiety weakens or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugation portion comprises a water-soluble polymer. In some embodiments, each water-soluble polymer independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each water-soluble polymer independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises polydextrose, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the water-soluble polymers each independently comprise a polyamine. In some embodiments, the conjugated moiety comprises a protein. In some embodiments, an additional conjugated moiety comprises a protein. In some embodiments, the proteins each independently comprise albumin, transferrin, or transthyretin. In some embodiments, the proteins each independently comprise an Fc moiety. In some embodiments, the proteins each independently comprise the Fc moiety of IgG. In some embodiments, the conjugated moiety comprises a peptide. In some embodiments, an additional conjugated moiety comprises a peptide. In some embodiments, the peptides each independently comprise XTEN peptides, high-amino acid polymers rich in glycine (HAP), PAS peptides, elastin-like peptides (ELP), CTP peptides, or gelatin-like protein (GLK) polymers. In some embodiments, the isolated and purified IL-2 peptide is modified by glutamylation. In some embodiments, the conjugated moiety binds directly to the isolated and purified IL-2 peptide. In some embodiments, the conjugated moiety binds indirectly to the isolated and purified IL-2 peptide via a linker. In some embodiments, the linker comprises a homologous bifunctional linker. In some embodiments, the homodifunctional linker comprises Lomant's reagent, dithiobis(succinimide propionate) DSP, 3'3'-dithiobis(succinimide propionate sulfonyl succinimide) (DTSSP), succinimide disuccinate (DSS), succinimide disuccinate (BS), succinimide tartrate (DST), disulfonyl tartrate (sulfonyl DST), glycosylbis(succinimide succinate) ethylene ester (EGS), glutarate succinimide (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl hexamethylenediimide (DMA), dimethyl heptamethine (DMP), dimethyl octanediimide (DMS), dimethyl-3,3'-dithiobispropionide (DSP) DTBP), 1,4-bis(3'-(2'-pyridyldithio)propamido)butane (DPDPB), bis(cis-butenyldiimide)hexane (BMH), compounds containing aryl halides (DFDNB) (e.g., 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the connector comprises a heterogeneous bifunctional connector. In some embodiments, the heterogeneous bifunctional connector comprises 3-(2-pyridyldithio)propionic acid N-succinimide ester (sPDP), long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (LC-sPDP), water-soluble long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (sulfonyl-LC-sPDP), succinimide-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), or sulfosuccinimide-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate. (Sulfon-LC-sMPT), succinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sulfon-sMCC), m-cis-butenyldiimide benzoyl-N-hydroxysuccinimide (MBs), m-cis-butenyldiimide benzoyl-N-hydroxysulfosuccinimide (sulfon-MBs), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB) ) sulfosuccinimide aminobenzoate (sulfon-sIAB), succinimide-4-(p-cis-butenylimide-phenyl)butyrate (sMPB), sulfosuccinimide-4-(p-cis-butenylimide-phenyl)butyrate (sulfon-sMPB), N-(γ-cis-butenylimide-butyryloxy)succinimide (GMBs), N-(γ-cis-butenylimide-butyryloxy)sulfosuccinimide (sulfon-GMBs), 6-((iodoacetyl)amino)hexanoic acid succinimide (sIAX), 6-[6-(((iodoacetyl)amino)hexanoic acid succinimide Acetyl)amino)hexanoyl)amino]hexanoic acid succinimide ester (sIAXX), 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylic acid succinimide ester (sIAC), 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoic acid succinimide ester (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl reactive and thiohydrogen-reactive crosslinking agents, such as 4-(4-N-cis-butenyldiimide-phenyl)butyric acid hydrazide (MPBH), 4-(N-cis-butenyldiimide-methyl)cyclohexane- 1-Carboxy-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfon-NHs-AsA), sulfosuccinimide-(4-azidosalicylamido)hexanoate (sulfon-NHs-LC-AsA), sulfosuccinimide-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimide-4 -Azide benzoate (HsAB), N-hydroxysulfosuccinimide-4-azidobenzoate (sulfon-HsAB), N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfon-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimide-2-(m-azido-o-nitrobenzoamide)-ethyl-1,3' -Dithiopropionate (sAND), N-succinimide-4-(4-azidophenyl)1,3'-dithiopropionate (sADP), (4-azidophenyl)-1,3'-dithiopropionate N-sulfosuccinimide (sulfon-sADP), 4-(p-azidophenyl)butyrate sulfosuccinimide (sulfon-sAPB), 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate sulfosuccinimide (sAED), 7-azido-4-methylcoumarin-3-acetic acid sulfosuccinimide (sulfon-s... AMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleic anhydride group, optionally comprising a maleic anhydride hexanoyl (mc), succinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sulfon-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives thereof, or the like. In some embodiments, the conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, an additional conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in this invention is polyethylene glycol-modified as disclosed in U.S. Patent Application Publication US 2020/0181220 A1 and U.S. Patent Application Publication US 2020/0330601 A1. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion relative to one residue of SEQ ID NO:5. In some embodiments, the IL-2 form suitable for the present invention lacks the IL-2Rα linker but maintains normal binding to the intermediate affinity IL-2Rβ-γ signaling complex. In some embodiments, the IL-2 form suitable for the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5.

In some embodiments, the IL-2 form suitable for use in this invention is Nevor interleukin-α (also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes). Nevo interleukin-α, also known as the human interleukin-2 fragment (1-59) variant (Cys ¹²⁵ > Ser ⁵¹ ), is fused to the human interleukin-2 fragment (62-132) via a peptide linker ( ⁶⁰ GG ⁶¹ ), which is fused to the human interleukin-2 receptor α chain fragment (139-303) via a peptide linker ( ¹³³ GSGGGS ¹³⁸ ). It is produced in Chinese hamster ovary (CHO) cells and is glycosylated. The human interleukin-2 (IL-2) (75-133)-peptide [Cys ¹²⁵ (51) > Ser]-mutant (1-59) is fused to the human interleukin-2 (IL-2) (4-74)-peptide (62-132) via a _G2 peptide linker (60-61) and via GSG _3. The S-peptide linker (133-138) is fused to the human interleukin 2 receptor α chain (IL2R subunit α, IL2Rα, IL2RA) (1-165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, in glycoform α. The amino acid sequence of Nevo interleukin α is provided in SEQ ID NO:6. In some embodiments, Nevo interleukin α exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbers in SEQ ID NO:6), and glycosylation sites at the following positions: N187, N206, T212 (using the numbers in SEQ ID NO:6). The preparation and properties of Nevo interleukin-α, as well as additional alternative forms of IL-2 suitable for the present invention, are described in U.S. Patent Application Publication No. US2021/0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the IL-2 form suitable for the present invention has the amino acid sequence provided in SEQ ID NO:6 or its conserved amino acid substitutions. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner via a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity with IL-1Rα and having receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises an immunoglobulin comprising all or part of an Fc region, wherein the mucin domain polypeptide linker comprises an amino acid sequence of SEQ ID NO:8 or having at least 90% sequence identity with SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

Table 2. Amino acid sequence of interleukin.

In some embodiments, the IL-2 form suitable for use in this invention comprises an antibody cytokine transplantation protein comprising: a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some implementations, the IL-2 regimen comprises administering the antibody described in U.S. Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine transplantation protein comprises: a heavy chain variable region (VH) including complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region (VL) including LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into a CDR of the _VH or _VL , wherein the IL-2 molecule is a mutant protein, wherein the antibody cytokine transplantation protein preferentially amplifies T effector cells relative to regulatory T cells, and wherein the antibody further comprises an IgG heavy chain and an IgG light chain selected from the group consisting of: an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:38; an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:29; an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:29; and an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:38.

In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR1 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR2 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR3 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR1 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR2 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR3 of _VL , wherein the IL-2 molecule is a mutant protein.

The insertion of an IL-2 molecule can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence replaces all or part of the CDR sequence. The IL-2 molecule replacement can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. The IL-2 molecule replacement can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

In some embodiments, the IL-2 molecule is directly grafted into a CDR without a peptide linker, wherein there are no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted into a CDR with a peptide linker, wherein there are one or more additional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecule described herein is an IL-2 mutant protein. In some cases, the IL-2 mutant protein contains an R67A substitution. In some embodiments, the IL-2 mutant protein contains the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutant protein contains the amino acid sequence of Table 1 in U.S. Patent Application Publication No. US 2020/0270334A1, the disclosure of which is incorporated herein by reference.

In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine transplantation protein comprises HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine transplantation protein comprises a _VH region containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine transplantation protein includes a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a light chain containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a _VH region containing the amino acid sequence of SEQ ID NO:28, and a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:38, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain region containing the amino acid sequence of SEQ ID NO:38; and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 or variants, derivatives, or fragments thereof, or conserved amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with them, as described in U.S. Patent Application Publication No. 2020/0270334A1. In some embodiments, the antibody component of the antibody cytokine transplantation protein described herein comprises the immunoglobulin sequence, framework sequence, or CDR sequence of palizumab. In some embodiments, the serum half-life of the antibody cytokine transplantation protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to aldehyde interleukin or similar molecules). In some embodiments, the antibody cytokine transplantation protein described herein has a sequence as illustrated in Table 3.

Table 3: Sequence of exemplary palizumab antibody-IL-2 transplanted protein

The term “IL-4” (also referred to herein as “IL4”) refers to a cytokine known as interleukin-4, produced by Th2 T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and _IgG1 expression. Recombinant human IL-4 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-211) and ThermoFisher Scientific of Waltham, MA, USA (recombinant human IL-15 protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for this invention is provided in Table 2 (SEQ ID NO:9).

The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine called interleukin-7, which is available from stromal and epithelial cells as well as dendritic cells. (Fry and Mackall, Blood 2002, 99, 3892-904). IL-7 stimulates T cell development. IL-7 binds to the IL-7 receptor (a heterodimer composed of IL-7 receptor α and a common γ chain receptor), which belongs to a family of signaling pathways important for T cell development within the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-254) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for this invention is provided in Table 2 (SEQ ID NO: 10).

The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor called interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the contents of which are incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-230-b) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number 34-8159-82). The amino acid sequence of the recombinant human IL-15 suitable for use in this invention is provided in Table 2 (SEQ ID NO: 11).

The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the contents of which are incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-408-b) and Thermo Fisher Scientific of Waltham, Massachusetts (recombinant human IL-21 protein, catalog number 14-8219-80). The amino acid sequence of the recombinant human IL-21 suitable for use in this invention is provided in Table 2 (SEQ ID NO:21).

When indicated as “effective antitumor dose,” “effective tumor-suppressing dose,” or “therapeutic dose,” the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease. Generally, it can be stated that the pharmaceutical composition described herein, comprising tumor-infiltrating lymphocytes (e.g., passaged TILs or genetically modified cytotoxic lymphocytes), can be administered at doses of ^10⁴ to ^10¹¹ cells/kg body weight (e.g., ^10⁵ to ^10⁶ , ^10⁵ to ^10¹⁰ , ^10⁵ to ^10¹¹ , ^10⁶ to ^10¹⁰ , ^{10⁶ to 10¹¹ , 10⁷} to ^10¹¹ , ^10⁷ to ^10¹⁰ , 10⁸ to ^10¹¹ , ^10⁸ to ^10¹⁰ , ^10⁹ to ^10¹¹ , or ^10⁹ to ^10¹⁰ cells/kg body weight), including all integer values within the range. TIL (in some cases, including genetically modified cytotoxic lymphocytes) compositions may also be administered at these doses multiple times. TILs (including genetically engineered TILs in some cases) can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). The optimal dosage and treatment regimen for a particular patient can be readily determined by medical professionals by monitoring the patient’s disease symptoms and adjusting the treatment accordingly.

The term "hematological malignancy" or related terms refer to cancers and tumors of the hematopoietic and lymphoid tissues of mammals (including, but not limited to, tissues of the blood, bone marrow, lymph nodes, and lymphatic system). Hematological malignancies are also known as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancy" refers to hematological malignancies affecting B cells.

The term "liquid tumor" refers to an abnormal clump of cells that is fluid in nature. Liquid tumor cancers include (but are not limited to) leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs derived from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs derived from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

As used herein, the term “microenvironment” can refer to the solid or hematologic tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanotransmitters that “promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, encourage treatment resistance, and provide a niche for the vigorous growth of dominant metastases,” as described by Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression of the microenvironment.

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (on days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m²/day for 5 days (on days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours to achieve physiological tolerance.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for components of the immune system (“cytokine pool”) prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as “immunosuppressive modulation”) in a patient prior to the introduction of the TIL of the present invention.

The term "effective amount" or "therapeutic effective amount" refers to an amount of compound or combination of compounds as described herein that is sufficient to achieve the intended application, including (but not limited to) the treatment of a disease. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo) or the subject being treated and the disease condition (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. The term also applies to doses that will induce a specific response in target cells (e.g., reduced platelet adhesion and/or cell migration). Specific doses will vary depending on: the specific compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

The terms “treatment”, “treating”, “treat”, etc., refer to achieving a desired pharmacological and/or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of partial or complete cure of a disease and/or adverse effects attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development or progression; and (c) alleviating the disease, i.e., causing disease remission and/or alleviating one or more disease symptoms. “Treatment” is also intended to cover the delivery of agents to provide a pharmacological effect, even in the absence of a disease or ailment. For example, “treatment” covers the delivery of compositions that can induce or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

The terms “non-myeloablative chemotherapy,” “non-myeloablative lymphocyte depletion,” “NMALD,” “NMA LD,” “NMA-LD,” and any variations thereof are used interchangeably to indicate a chemotherapy regimen designed to deplete a patient’s lymphoid immune cells while avoiding depletion of their myeloid immune cells. Typically, a patient receives one cycle of non-myeloablative chemotherapy before receiving tumor-infiltrating lymphocytes as described herein.

When used in conjunction with parts of a nucleic acid or protein, the term "heterologous" indicates that the nucleic acid or protein contains two or more subsequences that are not found in nature to be identical to each other. For example, nucleic acids are often produced in a recombinant manner, having two or more sequences from unrelated genes arranged to create new functional nucleic acid sequences, such as a promoter from one source and a coding region from another source or coding regions from different sources. Similarly, a heterologous protein indicates a protein that contains two or more subsequences that are not found in nature to be identical to each other (e.g., fusion proteins).

In the context of two or more nucleic acids or polypeptides, the terms "sequence identity," "percent identity," and "sequence percent identity" (or their synonyms, such as "99% identical") refer to two or more sequences or subsequences that are identical or have the same specific percentage of nucleotide or amino acid residues when compared and aligned (introducing gaps where necessary) to achieve maximum correspondence and without considering any conserved amino acid substitutions as part of the sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. Suitable programs for determining the percentage of sequence identity include, for example, the BLAST package available from the U.S. Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California, USA), or MegAlign (available from DNASTAR) are other publicly available software programs that can be used for sequence alignment. Those skilled in the art can determine the appropriate parameters for optimal alignment using specific alignment software. In some embodiments, the default parameters of the alignment software are used.

As used herein, the term "variant" covers (but is not limited to) antibodies or fusion proteins that contain an amino acid sequence different from that of a reference antibody, differing in that one or more substitutions, deletions, and/or additions are present within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conserved substitutions in its amino acid sequence compared to that of the reference antibody. Conserved substitutions may involve, for example, substitutions of similar charged or uncharged amino acids. Variants retain the ability to bind specifically to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

In this document, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are cells obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any population of expanded or proliferated TIL cells as discussed herein, including (but not limited to) the main TILs and expanded TILs (“REP TILs”) as discussed herein, as well as “reREP TILs”. reREP TILs may include, for example, a second expanded TIL or a second additional expanded TIL (e.g., the TILs described in step D of Figure 8, including TILs referred to as reREP TILs).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to be reintroduced into patients after infiltrating solid tumors. TILs can be further characterized by potency; for example, if interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, then TILs can be considered potent. TILs can be considered potent if, for example, interferon (IFNγ) releases greater than approximately 50 pg/mL, greater than approximately 100 pg/mL, greater than approximately 150 pg/mL, or greater than approximately 200 pg/mL, greater than approximately 300 pg/mL, greater than approximately 400 pg/mL, greater than approximately 500 pg/mL, greater than approximately 600 pg/mL, greater than approximately 700 pg/mL, greater than approximately 800 pg/mL, greater than approximately 900 pg/mL, or greater than approximately 1000 pg/mL.

The term "deoxyribonucleic acid" encompasses both natural and synthetic, unmodified and modified deoxyribonucleic acids. Modifications include alterations to the linkages between deoxyribonucleic acids in the sugar moiety, base moiety, and/or oligonucleotides.

The term "RNA" is defined as a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" is defined as a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, RNA produced in a recombinant manner, and altered RNA that differs from naturally occurring RNA through the addition, deletion, substitution, and/or modification of one or more nucleotides. The nucleotides in the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, as well as inert components. The use of such pharmaceutically acceptable carriers or excipients for the active pharmaceutical ingredient is well known in the art. Unless any conventionally pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is covered. Additional active pharmaceutical ingredients, such as those of other pharmaceuticals, may also be incorporated into the described compositions and methods.

The terms “about” and “approximately” mean a range of values that are statistically significant. This range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The permissible differences covered by the terms “about” or “approximately” depend on the specific system under study and are readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not precise and do not need to be precise, but may be approximate and/or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. Generally, whether so explicitly stated or not, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are “about” or “approximately”. It should be noted that embodiments with very different sizes, shapes, and dimensions may employ the described arrangement.

When used in the appended claims in both original and modified forms, the transitional terms “comprising,” “consisting essentially of,” and “consisting of” define the scope of the invention with respect to which additional, unrecited elements or steps (if present) are excluded from the scope of the claims. The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case excludes impurities generally associated with the specified materials. The term “consisting essentially of” limits the scope of the invention to the specified elements, steps, or materials and those elements, steps, or materials that do not substantially affect the basis and novel features of the claimed invention. In alternative embodiments, all compositions, methods, and kits for practicing the invention described herein may be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

The term “antibody” and its plural form “antibodies” refer to a complete immunoglobulin and any antigen-binding fragment (“antigen-binding moiety”) or its single chain. “Antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or its antigen-binding moiety. Each heavy chain includes a heavy chain variable region (abbreviated as _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated as _VL ) and a light chain constant region. The light chain constant region comprises one domain, _CL . The _VH and _VL regions of an antibody can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), and these may be interspersed with more conserved regions called frame regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more epitopes. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen" refers to a substance that induces an immune response. In some embodiments, if presented via a major histocompatibility complex (MHC) molecule, the antigen is a molecule capable of being bound by an antibody or TCR. As used herein, the term "antigen" also encompasses T-cell epitopes. Antigens are additionally capable of being recognized by the immune system. In some embodiments, the antigen is capable of inducing a humoral or cellular immune response that results in the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require the antigen to contain or be linked to a Th-cell epitope. The antigen may also have one or more epitopes (e.g., B epitopes and T epitopes). In some embodiments, the antigen is preferably to react generally with its corresponding antibody or TCR in a highly specific and selective manner, and not with a variety of other antibodies or TCRs that can be induced by other antigens.

The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to formulations of antibody molecules that are single molecular compositions. Monoclonal antibody compositions exhibit single binding specificity and affinity against a specific epitope. Monoclonal antibodies specific to certain receptors can be prepared using knowledge and techniques in the following methods: injecting a suitable antigen into a test subject and then isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. DNA encoding monoclonal antibodies can be isolated and sequenced using routine procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not normally produce immunoglobulins (e.g., *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to obtain monoclonal antibody synthesis in recombinant host cells. The recombinant production of antibodies will be described in more detail below.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Antigen-binding function of antibodies has been demonstrated to be performed by fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of _VL , _VH , _CL , and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; (iii) Fd fragments consisting of _VH and CH1 domains; (iv) Fv fragments consisting of _VL and _VH domains of a single arm of an antibody; (v) domain antibody (dAb) fragments (Ward et al., Nature, 1989, 341, 544-546), which may consist of a _VH or a _VL domain; and (vi) segregated complementarity-determining regions (CDRs). Furthermore, although the two domains ( _VL and _VH ) of the Fv fragment are encoded by independent genes, they can be joined using recombination methods via a synthetic adapter that enables the creation of a single protein chain (referred to as a single-chain Fv (scFv)) in which the _VL and _VH regions pair to form a monovalent molecule; see, for example, Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are targeted at the utility screening fragment in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises both the _VH and _VL portions. A scFv molecule is represented as _VL - _LVH when the _VL domain is the N-terminal portion of the scFv molecule, or as _VH - _LVL when the _VH domain is the N-terminal portion of the scFv molecule. Methods for preparing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB, Vol. 9: 73-80 (1995); and REBird and BWWalker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9: 132-137 (1991), the disclosures of which are incorporated herein by reference.

As used herein, the term "human antibody" is intended to include antibodies that have variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, said constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human framework sequence.

The term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity with a variable region, wherein both the framework and CDR regions of the variable region are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is generated from a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with immortalized cells, having a genome containing both human heavy-chain and light-chain transgenes.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas prepared therefrom (described further below) that are transgenic for the human immunoglobulin gene; (b) antibodies isolated from host cells transformed to express human antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may undergo in vitro mutation induction (or in vivo somatic mutation induction when using transgenic animals with human Ig sequences), and therefore the amino acid sequences of the _VH and _VL regions of the recombinant antibody, although derived from and associated with human germline _VH and _VL sequences, may not be naturally present in the human antibody germline in vivo.

As used in this article, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

The term "human antibody derivative" refers to any modified form of a human antibody, including conjugates of antibodies with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which may be conjugated to the antibody described herein using methods available in the art.

The terms "humanized antibody" and "humanized" refer to antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence. Further frame region modifications can be made to the human frame sequence. Humanized forms of non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. To a large extent, humanized antibodies are human immunoglobulins (receptor antibodies) where residues from the hypervariable region of the receptor are replaced by residues from 15 hypervariable regions of a non-human species (donor antibody) possessing the desired specificity, affinity, and ability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in receptor or donor antibodies. These modifications are performed to further optimize antibody performance. Generally, humanized antibodies will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially all FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein may also be modified to use any Fc variant known to provide effector function and/or FcR binding improvements (e.g., reductions). Fc variants may include any of the amino acid substitutions disclosed below: International Patent Application Publications No. WO 1988/07089A1, WO 1996/14339A1, WO 1998/05787A1, WO 1998/23289A1, WO 1999/51642A1, WO 99/58572A1, WO 2000/09560A2, WO 2000/32767A1, WO 20 No. 00/42072A2, No. WO 2002/44215A2, No. WO 2002/060919A2, No. WO 2003/074569 A2, No. WO 2004/016750 A2, No. WO 2004/029207 A2, No. WO 2004/035752 A2, No. WO 2004/063351A2, No. WO 2004/074455 A2, No. WO 2004/09924 9 A2, WO 2005/040217 A2, WO 2005/070963 A1, WO 2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 A1, WO 2006/047350 A2 and WO 2006/085967 A2; and U.S. Patent No. 5,648,260; Nos. 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the contents of which are incorporated herein by reference.

The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

A “bifunctional antibody” is a small antibody fragment having two antigen-binding sites. The fragment contains a heavy chain variable domain (V_{_H} ), which is linked to a light chain variable domain (V_L ) in the same polypeptide chain (V_{_H}-V_{_L} or V_{_L}-V_{_H}). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, thus creating two antigen-binding sites. Bifunctional antibodies are described in more detail, for example, in European Patent No. 404,097, International Patent Publication No. WO 93/11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

The term "glycosylation" refers to a modified derivative of an antibody. Deglycosylated antibodies are not glycosylated. Glycosylation can be altered to, for example, increase the antibody's affinity for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at said sites. Deglycosylation can increase the antibody's affinity for an antigen, as described in U.S. Patents 5,714,350 and 6,350,861. Alternatively or additionally, antibodies with altered glycosylation types can be produced, such as low-fucosylated antibodies with reduced fucosylation residues or antibodies with increased fractional GlcNac structure. Such altered glycosylation patterns have been shown to enhance antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention to produce glycosylated-modified antibodies. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)fucosyltransferase), so that antibodies expressed in Ms704, Ms705, and Ms709 cell lines do not have fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/- cell lines were formed by breaking the FUT8 gene in targeted CHO/DG44 cells using two alternative vectors (see, for example, U.S. Patent Publication No. 2004/0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation by reducing or eliminating α1,6 bond-related enzymes. The patent also describes cell lines with low or no enzymatic activity for adding fucose to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). International Patent Publication WO 03/035835 describes a variant CHO cell line, namely Lec 13 cells, which has a reduced ability to link fucose to Asn(297)-linked carbohydrates and also induces hypofucosylation of antibodies expressed in said host cells (see also Shields et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication WO 99/54342 describes cells engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine). Cell lines containing GnTIII transferase III (GnTIII) can be used to enhance the antibody's ADCC activity by causing antibodies expressed in engineered cell lines to exhibit an increased bipartite GlcNac structure (see also Umana et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidases can be used to cleave the fucosylate residues of the antibody. For example, α-L-fucosidase removes fucosylate residues from the antibody, as described in Tarentino et al., Biochem. 1975, 14, 5516-5523.

“Polyglycolization” refers to the modification of an antibody or fragment thereof, typically with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that link one or more PEG groups to the antibody or antibody fragment. For example, PEGylation can increase the biological (e.g., serum) half-life of an antibody. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to cover any form of PEG that has been used to derive other proteins, such as mono( _C1 - _C10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-cis-butenediamide. The antibody to be PEGylated is a deglycosylated antibody. Polyethylene glycolation methods are known in the art and applicable to antibodies used in this invention, such as those described, for example, in European Patents EP0154316 and EP0401384 and U.S. Patent No. 5,824,778, the disclosures of which are each incorporated herein by reference.

The term "biosimilar" refers to a biological product (including monoclonal antibodies or proteins) that meets the following criteria: it is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, and there are no clinically significant differences between the biological product and the reference product in terms of safety, purity, and efficacy. Furthermore, a biosimilar or "biosimilar" drug is a biological drug similar to another biological drug authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. A biological product or biological drug is a drug derived from or produced from a biological source (e.g., bacteria or yeast). It may consist of relatively small molecules (e.g., human insulin or erythropoietin) or complex molecules (e.g., monoclonal antibodies). For example, if the reference IL-2 protein is proleukin, then the protein of the reference proleukin approved by a drug regulatory agency is "biosimilar to proleukin" or "a biosimilar of proleukin." In Europe, a biosimilar or “biosimilar” medicine is a biological medicine similar to another biological medicine that has been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for the use of biosimilars in Europe is Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended, and therefore in Europe, biosimilars may be authorized, approved for authorization, or submitted as the subject of authorization pursuant to Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC. The original biological medicine product authorized in Europe may be referred to as a “reference medicine.” The CHMP guidance on biosimilar products outlines some of the requirements for a product to be considered a biosimilar. In addition, product-specific guidance, including guidance relating to monoclonal antibody biosimilars, is provided by the EMA on a product-by-product basis and published on its website. Biosimilars as described herein may be similar to a reference medicine in terms of quality characteristics, biological activity, mechanism of action, safety profile, and/or efficacy. Furthermore, biosimilars may be used or intended for the treatment of the same disease as the reference medicine. Therefore, biosimilars as described herein can be considered to have similar or very similar quality characteristics to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar biological activity to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar safety profile to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar efficacy to the reference drug. As described herein, in Europe, biosimilars have been compared with reference drugs authorized by the EMA. However, in some cases, in certain studies, biosimilars may be compared with biological products authorized outside the European Economic Area (non-EEA authorized "comparatives"). Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to biological products that have been or may be compared with non-EEA authorized comparatives. Some biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding moieties), and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications (including, for example, amino acid deletions, additions, and/or substitutions) in their amino acid structure that do not significantly affect peptide function. Biosimilars may contain an amino acid sequence having 97% or higher, such as 97%, 98%, 99%, or 100%, sequence identity with the amino acid sequence of a reference drug. Biosimilars may contain one or more post-translational modifications that differ from those of the reference drug, such as (but not limited to) glycosylation, oxidation, deamidation, and/or truncation, provided that such differences do not alter the safety and/or efficacy of the drug. Biosimilars may have the same or different glycosylation patterns as the reference drug. In particular, but not exclusively, biosimilars may have different glycosylation patterns if such differences resolve or are intended to resolve safety concerns associated with the reference drug. Additionally, biosimilars may differ from the reference drug in, for example, their potency, pharmaceutical form, formulation, excipients, and/or delivery method, provided that this does not impair the safety and efficacy of the drug. Compared to a reference product, biosimilars may contain differences in, for example, pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles, but are still considered sufficiently similar to the reference product and thus eligible for or suitable for authorization. In some cases, biosimilars exhibit different binding characteristics compared to the reference product, where the regulatory agency (e.g., the EMA) does not consider these different binding characteristics as an obstacle to authorization of the biosimilar product. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions.

II. Gene Editing Process

A. Overview: TIL amplification + gene editing

Embodiments of the present invention relate to methods for expanding a TIL population, said methods comprising one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, “gene-editing” and “genome editing” refer to a gene modification in which DNA is permanently modified in the genome of a cell, such as by inserting, deleting, modifying, or replacing DNA within the cell's genome. In some embodiments, gene editing causes silencing (sometimes referred to as gene knockout) or suppression/reduction (sometimes referred to as gene knockdown) of DNA sequence expression. According to embodiments of the present invention, gene editing technology is used to enhance the effectiveness of a therapeutic TIL population.

Methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the methods further include gene editing of at least a portion of the TILs. According to other embodiments, methods for expanding TILs into a therapeutic TIL population can be carried out according to any embodiment of the methods described in WO 2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, which are incorporated herein by reference in their entirety, wherein the methods further include gene editing of at least a portion of the TILs. Therefore, one embodiment of the invention provides a therapeutic TIL population expanded according to any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, for example, at least a portion of the therapeutic TIL population transferred to an infusion bag has been permanently gene-edited.

Gene editing during B.TIL amplification

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the method includes the step of culturing or initially expanding a first TIL population, comprising culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some embodiments, the method includes the steps of culturing or rapidly expanding the third TIL population by culturing the third TIL population in a second cell culture medium for a first period of about 1-7 days, splitting the culture into multiple subcultures at the end of the first period, culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 3-7 days, and merging the multiple subcultures at the end of the second period to provide an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the activation of the second TIL population takes approximately 7 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during, or before, any of steps (a)-(e), (a)-(f), or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e), (a)-(f), or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A third population of TILs was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some embodiments, the step of culturing the third TIL population takes approximately 7 days. In some embodiments, the step of culturing the third TIL population takes approximately 8 days. In some embodiments, the step of culturing the third TIL population takes approximately 9 days. In some embodiments, the step of culturing the third TIL population takes approximately 10 days. In some embodiments, the step of culturing the third TIL population takes approximately 11 days. In some embodiments, the step of culturing the third TIL population takes approximately 12 days. In some embodiments, the step of culturing the third TIL population takes approximately 13 days. In some embodiments, the step of culturing the third TIL population takes approximately 14 days. In some embodiments, the step of culturing the third TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(d) or (a)-(e) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(d) or (a)-(e), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some implementation schemes, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementation schemes, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are then placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 1-7 days. In some implementations, the step of culturing the third TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the third TIL population takes approximately 1 day. In some implementations, the step of culturing the third TIL population takes approximately 2 days. In some implementations, the step of culturing the third TIL population takes approximately 3 days. In some implementations, the step of culturing the third TIL population takes approximately 4 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some implementations, the process of culturing a third TIL population takes about 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed over a period of approximately 22 days. In some embodiments, the steps of the method are completed over a period of approximately 23 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some implementations, the steps of the method are completed over a period of approximately 22 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1 day. In some implementations, the step of culturing the fourth TIL population takes approximately 2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 3 days. In some implementations, the step of culturing the fourth TIL population takes approximately 4 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, initial amplification takes approximately 3-9 days. In other implementations, initial amplification takes approximately 1-9 days, 2-9 days, 3-9 days, approximately 4-9 days, approximately 5-9 days, approximately 6-9 days, approximately 7-9 days, approximately 8-9 days, approximately 1-8 days, approximately 2-8 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, initial amplification takes approximately 1 day. In some embodiments, initial amplification takes approximately 2 days. In some embodiments, initial amplification takes approximately 3 days. In some embodiments, initial amplification takes approximately 4 days. In some embodiments, initial amplification takes approximately 5 days. In some embodiments, initial amplification takes approximately 6 days. In some embodiments, initial amplification takes approximately 7 days. In some embodiments, initial amplification takes approximately 8 days. In some embodiments, initial amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the rapid second amplification takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error or incomplete sentence.) The durations of rapid secondary amplification are approximately 11-13 days, 12-13 days, 5-12 days, 6-12 days, 7-12 days, 8-12 days, 9-12 days, 10-12 days, 11-12 days, 5-11 days, 6-11 days, 7-11 days, 8-11 days, 9-11 days, 10-11 days, 5-10 days, 6-10 days, 7-10 days, 8-10 days, 9-10 days, 5-9 days, 6-9 days, 7-9 days, 8-9 days, 5-8 days, 6-8 days, 7-8 days, 5-7 days, 6-7 days, and 5-6 days. In some implementations, rapid secondary amplification takes approximately 5 days. In some implementations, rapid secondary amplification takes approximately 6 days. In some implementations, rapid secondary amplification takes approximately 7 days. In some embodiments, rapid second amplification takes approximately 8 days. In some embodiments, rapid second amplification takes approximately 9 days. In some embodiments, rapid second amplification takes approximately 10 days. In some embodiments, rapid second amplification takes approximately 11 days. In some embodiments, rapid second amplification takes approximately 12 days. In some embodiments, rapid second amplification takes approximately 13 days. In some embodiments, rapid second amplification takes approximately 14 days. In some embodiments, rapid second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digest the tumor tissue sample or tumor fragment in an enzyme medium to produce tumor digest;

(c) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some implementations, the first amplification takes approximately 3-9 days. In some implementations, the first amplification takes approximately 3-9 days, approximately 3-8 days, approximately 3-7 days, approximately 3-6 days, approximately 3-5 days, approximately 3-4 days, approximately 4-9 days, approximately 4-8 days, approximately 5-9 days, approximately 5-8 days, approximately 6-9 days, approximately 6-8 days, approximately 7-9 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the first amplification takes approximately 3 days. In some implementations, the first amplification takes approximately 4 days. In some implementations, the first amplification takes approximately 5 days. In some implementations, the first amplification takes approximately 6 days. In some implementations, the first amplification takes approximately 7 days. In some implementations, the first amplification takes approximately 8 days. In some implementations, the first amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes approximately 5-15 days. In some implementations, the second amplification takes approximately 5-15 days, approximately 6-15 days, approximately 7-15 days, approximately 8-15 days, approximately 9-15 days, approximately 10-15 days, approximately 11-15 days, approximately 12-15 days, approximately 13-15 days, approximately 14-15 days, approximately 5-14 days, approximately 6-14 days, approximately 7-14 days, approximately 8-14 days, approximately 9-14 days, approximately 10-14 days, approximately 11-14 days, approximately 12-14 days, approximately 13-14 days, approximately 5-13 days, approximately 6-13 days, approximately 7-13 days, approximately 8-13 days, approximately 9-13 days, approximately 10-13 days. The durations of amplification are as follows: approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the second amplification takes approximately 5 days. In some implementations, the second amplification takes approximately 6 days. In some implementations, the second amplification takes approximately 7 days. In some implementations, the second amplification takes approximately 8 days. In some embodiments, the second amplification takes approximately 9 days. In some embodiments, the second amplification takes approximately 10 days. In some embodiments, the second amplification takes approximately 11 days. In some embodiments, the second amplification takes approximately 12 days. In some embodiments, the second amplification takes approximately 13 days. In some embodiments, the second amplification takes approximately 14 days. In some embodiments, the second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days. In some embodiments, the steps of the method are completed within a period of approximately 32 days.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

In some implementations, gene editing is performed while the TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementations, TILs collected from the culture medium are gene-edited, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, the step of gene editing at least a portion of the second or third TIL population includes a sterile electroporation step on the second or third TIL population.

In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some embodiments, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step further includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 22 hours. According to some implementations, the quiescent step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the antigen-presenting cells (APCs) are PBMCs. According to some embodiments, the PBMCs are irradiated. According to some embodiments, the PBMCs are allogeneic. According to some embodiments, the PBMCs are both irradiated and allogeneic. According to some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some implementations, the tumor tissue is derived from an autopsied tumor.

In some implementations, the dissected tumor is less than 8 hours old.

In some embodiments, the tumor tissue is selected from the group consisting of: melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple-negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and HPV-positive tumor tissue.

In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 2 mm.

In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 1.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5.5 mm and a longest side length of about 6 mm.

In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm or about 6 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 6 mm.

In some embodiments, the present invention provides a therapeutic population of tumor-infiltrating lymphocyte (TIL) products generated by methods as described herein.

In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising administering to the patient an effective amount of a therapeutic TIL population generated by the method described herein. In some embodiments, the cancer is selected from the group consisting of: glioblastoma (GBM), gastrointestinal cancer, melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, and mantle cell lymphoma. In some implementations, the cancer is selected from the group consisting of: cutaneous melanoma, ocular melanoma, uveal melanoma, conjunctival malignant melanoma, metastatic melanoma, pleomorphic xanthoastrocytoma, dysembryonic neuroepithelial tumor, ganglioglioma and pilocytic astrocytoma, endometrioid adenocarcinoma with significant myxoid differentiation (ECMD), papillary thyroid carcinoma, serous low-grade or borderline ovarian cancer, hairy cell leukemia and Langerhans cell histiocytosis.

In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 2000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 2000 IU/mL.

In some embodiments, in the second amplification step, the IL-2 is present at an initial concentration between 1000 IU/mL and 6000 IU/mL, and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some embodiments, the first amplification is performed using a ventilated container. In some embodiments, the second amplification is performed using a ventilated container.

In some embodiments, the first cell culture medium further comprises cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and/or the third culture medium further comprise cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the method further includes the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated the day after administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated on the same day as administration of the TIL or PBL product. In some embodiments, the IL-2 regimen comprises adelleukin, nevoleukin, or a bioanalyte or variant thereof.

In some embodiments, the therapeutically effective amount of TIL product comprises about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs.

In some implementations, the number of the second TIL population is at least 50 times that of the first TIL population.

C.PD-1

One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 superfamily of T cell regulators. Its ligands, PD-L1 and PD-L2, are expressed on various tumor cells, including melanoma. The interaction between PD-1 and PD-L1 can suppress T cell effector function, cause T cell exhaustion under chronic stimulation, and induce T cell apoptosis in the tumor microenvironment. PD-1 may also play a role in tumor-specific evasion of immune surveillance.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of PD-1 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the method includes gene editing of at least a portion of the TILs by silencing or inhibiting the expression of PD-1. As described in more detail below, the gene editing process may involve the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., PD-1). For example, the TALEN method can be used to silence or reduce the expression of PD-1 in TILs.

D.CTLA-4

CTLA-4 expression is induced upon T cell activation on activated T cells and competitively binds to antigen-presenting cell activation antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 can induce T cell suppression and is used to maintain the homeostasis of the immune response. However, inhibiting the interaction of CTLA-4 with CD80 or CD86 prolongs T cell activation and thus increases the level of the immune response against cancer antigens.

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of CTLA-4 in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and CTLA-4 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CTLA-4. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CTLA-4). For example, the CRISPR method, the TALE method, or the zinc finger method can be used to silence or inhibit the expression of CTLA-4 in TILs. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and CTLA-4 in TILs.

E.LAG-3

Following conjugation at the major histocompatibility complex (MHC) class II, lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells. Although the mechanism is not fully understood, regulation of this gene can induce negative regulation of T cell function, preventing tissue damage and autoimmunity. Therefore, LAG-3 blockade may improve antitumor responses. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of LAG-3 in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and LAG-3 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of LAG-3. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., LAG-3). According to specific embodiments, the expression of LAG-3 in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and LAG-3 in TILs can be silenced or reduced using the TALEN method.

F.Cish

Cish, a member of the cytokine signaling inhibitory factor (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits its functional affinity for tumors. Deletion of the Cish gene in CD8+ T cells enhances its amplification, functional affinity, and cytokine pluripotency, leading to significant and durable regression of existing tumors. See, for example, Palmer et al., Journal of Experimental Medicine, 212(12):2095 (2015).

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of Cish in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and Cish in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of Cish. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., Cish). For example, the expression of Cish in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and Cish in TILs can be silenced or reduced using the TALEN method.

G.CBL-B

CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and a negative regulator of T cell activation. (Bachmaier et al., Nature, 2000, 403, 211-216; Wallner et al., Clin. Dev. Immunol. 2012, 692-639.)

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of CBL-B in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and CBL-B in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., process 2A or the method shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CBL-B. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CBL-B). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, CBL-B is silenced using TALEN gene knockout. In some embodiments, CBL-B is silenced using a TALE-KRAB transcription inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and CBL-B in TIL.

H.TIGIT

TIGIT is a cell surface protein expressed on regulatory, memory, and activated T cells. TIGIT belongs to the poliovirus receptor (PVR) family of immunoglobulins and inhibits T cell activation. (Yu et al., Nat Immunol., 2009, 10(1):48-57).

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of TIGIT in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and TIGIT in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of TIGIT. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., TIGIT). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, TIGIT can be silenced using TALEN gene knockout. In some embodiments, TIGIT can be silenced using a TALE-KRAB transcriptional inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and TIGIT in TIL.

III. Gene Editing Methods

As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) genetically modified via gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing performed via nucleotide insertion (RNA or DNA) into a TIL population to suppress the expression of one or more proteins. Embodiments of the present invention also provide a method for expanding TILs into a therapeutic population, wherein the method includes gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, and these gene editing techniques are suitable for use according to the present invention.

In some embodiments, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes a retroviral transduction step. In one embodiment, the method of genetically modifying a TIL population includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, a method for genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which a transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long-term expression of the transposase does not occur in transgenic cells, for example, a transposase provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems include salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In one embodiment, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing or repressing (e.g., silencing) one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In another embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating TILs with a pulsed electric field to induce pore formation in the TILs, including the step of applying a series of at least three DC electrical pulses to the TILs, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time and such that the survival rate of the TILs is maintained. In one embodiment, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes the step of transfection using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

According to one embodiment, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems, such as Cas9, target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA bases and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, a method for amplifying TILs into a therapeutic population may be performed according to any embodiment of the method described herein (e.g., procedure 2A) or as described in WO2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, wherein said method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by comparing gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

1. TALE method

A method for amplifying TILs into a therapeutic population can be made according to any embodiment of the method described herein or as described in WO 2018/081473 A1, WO 2018/129332A1, or WO 2018/182817 A1, wherein the method further includes gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the use of the TALE method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the TALE method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for “transcription activator-like effector” protein, which includes TALEN (“transcription activator-like effector nuclease”). The method of gene editing using the TALE system is also referred to herein as the TALE method. TALE is a naturally occurring protein from the plant pathogenic bacterium *Xanthomonas* and contains a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize continuous DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by 14–20 base-pair spacers, bring FokI monomers closer together for dimerization and to generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and T NFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA , IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

Non-limiting examples of TALE-nucleases targeting the PD-1 gene are provided in the table below. In these examples, the targeted genomic sequence contains two 17-base-pair (bp) long sequences (referred to as half-targets, shown in uppercase letters) separated by a 15-bp spacer. Each half-target is recognized by a repetitive sequence of the half-TALE-nuclease listed in the table. Thus, according to a particular embodiment, the TALE-nuclease according to the invention recognizes and cleaves target sequences selected from the group consisting of: SEQ ID NO:127 and SEQ ID NO:128. TALEN sequences and gene editing methods are also described in Gautron et al., Molecular Therapy: Nucleic Acids, December 2017, Vol. 9: 312-321, which is incorporated herein by reference.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

2. Cas-CLOVER method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs using a Cas-CLOVER method. According to a particular embodiment, the use of the Cas-CLOVER method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the Cas-CLOVER method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Cas-CLOVER is a dimerizing, high-fidelity site-specific nuclease (SSN) composed of a fusion of catalytically dead SpCas9 (dCas9) and a nuclease domain from a Clostridium Clo051 type IIs restriction endonuclease (Madison et al., “Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched alloeneic CAR-T cells”, Molecular Therapy-Nucleic Acids, 2022). This produces a nuclease whose activity is determined by the dimerization of the Clo051 nuclease domain, which recognizes two adjacent 20-nt target sequences via RNA guidance. Unlike paired nicking enzyme approaches, for example, when using the Cas9-D10A mutant, monomeric Cas-CLOVER does not introduce nicks or DSBs. Cas-CLOVER has been shown to have low off-target nuclease activity.

Exemplary Cas-CLOVER systems include those described in WO2019/126578, the contents of which are incorporated herein by reference in their entirety. In embodiments, the Cas-CLOVER system comprises a fusion protein containing a DNA-localizing component and an effector molecule, substantially consisting of or consisting of therein.

DNA positioning components

In this embodiment, the DNA targeting component is capable of binding to a specific DNA sequence. In this embodiment, the DNA targeting component is selected from, for example, DNA-binding oligonucleotides, DNA-binding proteins, DNA-binding protein complexes, and combinations thereof. Other suitable DNA-binding components will be recognized by those skilled in the art.

In one embodiment, the DNA targeting component comprises oligonucleotides targeting one or more specific loci in the genome. The oligonucleotides may be selected from DNA, RNA, DNA/RNA hybrids, and combinations thereof.

In some embodiments, the DNA positioning component comprises a nucleotide-binding protein or protein complex that binds an oligonucleotide upon binding to the target DNA. The protein or protein complex may be able to recognize features selected from RNA-DNA heteroduplexes, R-loops, or combinations thereof. In some embodiments, the DNA positioning component comprises a protein or protein complex capable of recognizing an R-loop selected from Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, or combinations thereof. In some embodiments, the DNA positioning component comprises an engineered protein capable of binding to the target DNA. In some embodiments, the DNA positioning component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, transcription activator-like (TAL) arrays, and combinations thereof. In some embodiments, the DNA positioning component comprises a protein containing a naturally occurring DNA-binding domain. In some embodiments, the DNA positioning component comprises a bZIP domain, a helix-loop-helix, a helix-turn-helix, an HMG-box, a leucine zipper, a zinc finger, or combinations thereof. In some embodiments, the DNA positioning component comprises an oligonucleotide targeting a specific locus in the genome. Exemplary oligonucleotides include, but are not limited to, DNA, RNA, DNA/RNA hybrids, and any combination thereof. In some embodiments, the DNA targeting component comprises a protein or protein complex capable of recognizing features selected from RNA-DNA heteroduplexes, R-loops, and any combinations thereof. Exemplary proteins or protein complexes capable of recognizing R-loops include, but are not limited to, Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, and any combinations thereof. In some embodiments, the protein or protein complex capable of recognizing R-loops comprises Cas9. In some embodiments, the DNA targeting component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, TAL arrays, and any combinations thereof. In some embodiments, the DNA targeting component comprises an oligonucleotide targeting a specific location in the genome and a protein capable of binding to a target DNA sequence.

In one embodiment, the DNA positioning component comprises at least one guide RNA (gRNA), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises two gRNAs, substantially consisting of or consisting of them, wherein the first gRNA specifically binds to the first strand of the double-stranded DNA target sequence, and the second gRNA specifically binds to the second strand of the double-stranded DNA target sequence. Alternatively, in one embodiment, the DNA positioning component comprises a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Ralstonia*, substantially consisting of or consisting of it.

Effect molecules

In the implementation scheme, the effector molecule is capable of producing a predetermined effect at a specific locus in the genome. Exemplary effector molecules include, but are not limited to, transcription factors (activators or repressors), chromatin remodeling factors, nucleases, exonucleases, endonucleases, transposases, methyltransferases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, helicases, fluorophores, or any combination thereof.

In some embodiments, the effector molecule comprises a transposase. In some embodiments, the effector molecule comprises PB transposase (PBase). In some embodiments, the effector molecule comprises a nuclease. Non-limiting examples of nucleases include restriction endonucleases, homing endonucleases, S1 nucleases, mung bean nucleases, pancreatic DNase I, micrococcal nucleases, yeast HO endonucleases, or any combination thereof. In some embodiments, the effector molecule comprises a restriction endonuclease. In some embodiments, the effector molecule comprises a type IIS restriction endonuclease. In some embodiments, the effector molecule comprises a nuclease. Non-restrictive examples of endonucleases include AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, and Clo051. In the implementation scheme, the effector molecule includes BmrI, BfiI, or Clo051.

In embodiments, the effector molecule comprises a homodimer or heterodimer, substantially composed of, or composed of. In embodiments, the effector molecule comprises a nuclease, optionally an endonuclease, substantially composed of, or composed of. In embodiments, the effector molecule, including those comprising a homodimer or heterodimer, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially composed of, or composed of. In embodiments, Cas9 is catalytically inactive or “inactivated” Cas9 (dCas9 (SEQ ID NO: 302 and 303 of WO2019/126578)). In embodiments, Cas9 is the catalytically inactive or “inactivated” nuclease domain of Cas9. In embodiments, dCas9 is encoded by a shorter sequence derived from full-length, catalytically inactivated Cas9, referred herein as “small” dCas9 or dSaCas9 (SEQ ID NO: 23 of WO2019/126578).

In embodiments of the fusion protein, the effector molecule comprises, is substantially composed of, or is composed of, one or more type II nucleases, homodimers or heterodimers of type II nucleases. In the implementation scheme, the type II nuclease includes one or more of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof that forms a heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises a catalytically inactive form of Cas9 (e.g., dCas9 or dSaCas9) or a fragment thereof that forms a heterodimer with Clo051, and is substantially composed of or constitutes thereof. An exemplary Clo051 nuclease domain may comprise the following amino acid sequence, and is substantially composed of or constitutes thereof:

EGIKSNISLLKDELRGQISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE

KIRSGEMTIEELERAMFNNSEFILKY(SEQ ID NO:238).

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces*, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, and is substantially composed of or constitutes thereof.

connect

In some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule, substantially consisting of or consisting of the latter. In some embodiments, nucleic acid sequences encoding one or more components of the fusion protein can be operatively linked, for example, to an expression vector. In some embodiments, the fusion protein is a chimeric protein. In some embodiments, the fusion protein is encoded by one or more recombinant nucleic acid sequences. In some embodiments, the fusion protein further includes a linker region to operatively link the two components of the fusion protein. For example, in some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule operatively linked by a linker region, substantially consisting of or consisting of the latter. In some embodiments, the DNA positioning component, linker region, and effector molecule can be encoded by one or more nucleic acid sequences inserted into an expression cassette and/or expression vector, such that translation of the nucleic acid sequences produces the fusion protein. In some embodiments, the fusion protein can include a non-covalent link between the DNA positioning component and the effector molecule. The non-covalent link can comprise an antibody, antibody fragment, antibody mimic, or scaffold protein.

Fusion protein

In one embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated Cas9 (dCas9) or an inactivated nuclease domain, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated small Cas9 (dSaCas9), substantially consisting of or consisting of it. In yet another embodiment, the effector molecule comprises Cas9, dCas9, dSaCas9, or a nuclease domain thereof and a second endonuclease, substantially consisting of or consisting of it. The second endonuclease may include, or be substantially composed of, type IIS endonucleases, including but not limited to, AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, and BmgBI. One or more of BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI, or Clo051.

In embodiments of the fusion protein, the DNA localization component comprises, is substantially composed of, or is composed of, a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), and the effector molecule comprises, substantially composed of, or is composed of, a nuclease. In embodiments of the fusion protein of this disclosure, the DNA localization component comprises, substantially composed of, or is composed of, a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rhodotorula*, and the effector molecule comprises, substantially composed of, or is composed of, an IIS-type nuclease, including but not limited to AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My One or more of I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In some embodiments, the exemplary dCas9-Clo051 fusion protein may comprise, substantially consist of, or consist of the amino acid sequence of SEQ ID NO:305 or 307 of WO2019/126578 or the nucleic acid sequence of SEQ ID NO:306 or 308 of WO2019/126578.

Construct

In the embodiments, the nuclease domain comprises dCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises dSaCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Xanthomonas spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Rocheborax spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the fusion protein comprises dCas9-Clo051, dSaCas9-Clo051, Xanthomonas spp.-TALE-Clo051, or Rocheborax spp.-TALE-Clo051. In the embodiments, the vector encoding the fusion protein comprises Csy4-T2A-Clo051-G4S linker-dCas9 (Streptococcus pyogenes) or pRT1-Clo051-dCas9 dual NLS.

According to some implementations, the Cas-CLOVER system comprises a fusion protein containing a DNA localization component and an effector molecule, wherein the DNA localization component hybridizes to a target sequence of a DNA molecule in a TIL, wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule, and the effector molecule cleaves the DNA molecule, thereby altering the expression of at least one immune checkpoint molecule.

According to a specific implementation, the Cas-CLOVER method involves silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas-CLOVER system (e.g., dCas9-Clo051, dSaCas9-Clo051, Xanthomonas-TALE-Clo051, or Rochetomyces-TALE-Clo051 fusion protein) that is specific to the target DNA sequence of immune checkpoint genes. The fusion protein can be delivered as DNA, mRNA, or protein. After the genome comes into contact with the Cas-CLOVER system, one or more strands of the target double-stranded DNA can be cleaved. If cleavage occurs in the presence of one or more DNA repair pathways or components thereof, the Cas-CLOVER method interrupts gene expression or modifies the genomic sequence by inserting, deleting, or substituting one or more base pairs. DSBs in cells can be repaired via non-homologous end joining (NHEJ, a mechanism that typically causes insertions or deletions (insertion/deletion) in DNA). Insertions/deletions typically induce frameshifts, resulting in loss-of-function alleles; for example, by inducing premature stop codons within the open reading frame (ORF) of a target gene. According to some implementations, the result is a loss-of-function mutation within a target immune checkpoint gene.

Alternatively, instead of NHEJ, DSB induced by the Cas-CLOVER system can be repaired via homology-directed repair (HDR). Although NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to produce specific nucleotide changes ranging from a single nucleotide change to a large insertion range. According to some implementations, HDR is used to gene edit immune checkpoint genes by delivering a DNA repair template containing the desired sequence to a TIL equipped with a Cas-CLOVER system. The repair template preferably contains the desired edit immediately upstream and downstream of the target gene, as well as other homologous sequences (often referred to as left and right homologous arms).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the Cas-CLOVER method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF1. 0A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, I L6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via the Cas-CLOVER method and which can be used according to embodiments of the invention are described in WO2019126578, US2017/0107541, US2017/0114149, US2018/0187185, and U.S. Patent No. 10,415,024, the contents of which are incorporated herein by reference in their entirety. Resources for performing the Cas-CLOVER method, such as CLOVER mRNA and Cas-CLOVER mRNA constructs, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system regulating the expression of at least one checkpoint protein in multiple cells of a second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culture for about 1 to 3 days to obtain the second TIL population, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system inhibiting the expression of at least one checkpoint protein in multiple cells of the second TIL population.

IV. Gene Expression Methods

In some embodiments, the method of genetically modifying TIL populations to regulate protein expression may optionally include a step of stabilizing and incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying TIL populations includes a viral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a gamma-retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adenovirus transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adeno-associated virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a herpes simplex virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a poxvirus transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step, including lentiviral transduction using human immunodeficiency virus (HIV) (including HIV-1). Lentiviral transduction systems and other suitable viral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat’l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71, and U.S. Patents 5,350,674, 5,585,362, and 6,627,442, the disclosures of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16; Hawley et al., Gene Ther. 1994, 1, 136-38; the contents of which are incorporated herein by reference.

1. piggyBac method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs by a piggyBac method (e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases). According to a particular embodiment, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population. Alternatively, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population and optionally induce the enhancement of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. In some embodiments, at least one immunomodulatory composition comprises an immunomodulator fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and CD40 agonists (e.g., CD40L or agonistic CD40 binding domains). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and CD40 agonists. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and CD40 agonists.

The piggyBac transposon is a mobile genetic element that efficiently translocates between the donor vector and the host chromosome. This system has virtually no cargo limitations and is completely reversible, leaving no footprint in the genome after excision. The piggyBac transposon/transposase system consists of transposases that recognize piggyBac-specific inverted terminal repeats (ITRs) located flanking the transposon cassette. The transposases excise the transposable element to integrate it into a TT/AA chromosomal locus that preferentially resides in euchromatin regions of the mammalian genome (Ding et al., 2005; and Bradley 2007; Wilson et al., 2007; Wang et al., 2008; Li et al., 2011).

Exemplary piggyBac systems include those described in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac system comprises a transposon/transposase system.

In the implementation scheme, the piggyBac method includes delivering to the TIL a (a) nucleic acid or amino acid sequence containing a sequence encoding a transposase and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon.

In one embodiment, the sequence encoding the transposase is an mRNA sequence. In another embodiment, the sequence encoding the transposase is a DNA sequence. In yet another embodiment, the DNA sequence is a cDNA sequence. In yet another embodiment, the sequence encoding the transposase is an amino acid sequence. The protein Superpiggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA.

Transposons/transposases

Exemplary transposon/transposase systems include, but are not limited to, piggyBac transposon and transposase, Sleeping Beauty transposon and transposase, Helraiser transposon and transposase, and Tol2 transposon and transposase.

The piggyBac transposon recognizes the transposon-specific inverted terminal repeat (ITR) at the end of the transposon and moves the contents between the ITRs to the TTAA chromosomal locus. The piggyBac transposon system has no effective load limitation on the gene of interest that can be included between the ITRs. In embodiments, the transposon is a piggyBac transposon or a piggyBac-like transposon.

Examples of piggyBac and piggyBac-like transposases and transposons include, for example, those disclosed in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is overactive. An overactive piggyBac or piggyBac-like transposase is a transposase that is more active than its naturally occurring variants. In embodiments, the overactive piggyBac or piggyBac-like transposase is isolated from or derived from the silkworm (Bombyxmori). A list of overactive amino acid substitutions is available in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is an integration-deficient type. In embodiments, an integration-deficient piggyBac or piggyBac-like transposase is a transposase capable of cleaving its corresponding transposon but integrating the cleaved transposon at a lower frequency than the corresponding wild-type transposase. A list of defective amino acid substitutions can be found in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

In the embodiments, the piggyBac or piggyBac-like transposon can be inserted into the 5'-TTAT-3 sequence within the target nucleic acid via a piggyBac or piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or SuperpiggyBac ^™ (SPB) transposase. In the embodiments, and particularly in embodiments where the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

The Sleeping Beauty (SB) transposon is translocated into the target genome via a Sleeping Beauty transposase that recognizes an ITR, and moves the contents between ITRs to the TA chromosomal locus. In an embodiment, the transposon is a Sleeping Beauty transposon. In an embodiment, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In an embodiment, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase.

The Helraiser transposon is translocated by the Helitron transposase. Unlike other transposases, the Helitron transposase does not contain an RNase-H-like catalytic domain, but instead contains a RepHel motif consisting of a replication initiation domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH nuclease superfamily. In embodiments, the transposon is the Helraiser transposon. In embodiments of the Helraiser transposon sequence, the transposase is flanked by left and right terminal sequences, referred to as the LTS and RTS. In embodiments, these sequences terminate with a conserved 5'-TC/CTAG-3' motif. In embodiments, a 19 bp palindromic sequence with the potential to form a hairpin termination structure is located 11 nucleotides upstream of the RTS and contains the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG. In embodiments, and particularly in embodiments where the transposon is the Helraiser transposon, the transposase is the Helitron transposase.

The Tol2 transposon can be isolated from or derived from the genome of the killifish and can be similar to transposons of the hAT family. An exemplary Tol2 transposon of this disclosure is encoded by a sequence comprising approximately 4.7 kilobases and contains a gene encoding a Tol2 transposase, said gene containing four exons. In embodiments, the transposon is a Tol2 transposon. In some embodiments of the methods of this disclosure, and particularly in those embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

In some embodiments, the vector comprises a recombinant and non-naturally occurring DNA sequence encoding a transposon. In some embodiments, the vector comprises DNA of any form, and wherein the vector comprises at least 100 nucleotides (nt), 500 nt, 1000 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6500 nt, 7000 nt, 7500 nt, 8000 nt, 8500 nt, 9000 nt, 9500 nt, 10,000 nt, or any number of nucleotides in between. In some embodiments, the vector comprises single-stranded or double-stranded DNA. In some embodiments, the vector comprises circular DNA. In some embodiments, the vector is a plasmid vector, a nanoplasmid vector, or a small loop. In some embodiments, the vector comprises linear or linearized DNA. In some embodiments, the vector is a double-stranded doggybone ^™ DNA sequence.

In the implementation scheme, the recombinant and non-naturally occurring DNA sequences encoding transposons also include sequences encoding one or more immune checkpoint genes.

In the implementation scheme, the nucleic acid sequence encoding the transposase is a DNA sequence, and the amount of the DNA sequence encoding the transposase and the DNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the DNA sequences encoding transposases and the DNA sequences encoding transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation scheme, the nucleic acid sequence encoding the transposase is an RNA sequence, and the amount of the RNA sequence encoding the transposase and the RNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the RNA sequences encoding transposases and transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation, the TIL is further modified by a second gene-editing tool, including but not limited to those described herein. In the implementation, the second gene-editing tool may include a piggyBac transposase that is only excised to re-excise the inserted sequence or any portion thereof. For example, a piggyBac transposase that is only excised can be used to "re-excise" a transposon.

According to some embodiments, the piggyBac system comprises a transposon/transposase system, wherein the transposase recognizes ITRs located flanking a transposon cassette containing a cargo encoding one or more immune checkpoint genes, and excises transposable elements to integrate it into a TT/AA chromosomal site, resulting in genomic insertion of the transposon cassette and expression of one or more immune checkpoint genes. According to some embodiments, the cargo encodes two or more immune checkpoint molecules.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the piggyBac method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD), and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using the piggyBac method and which can be used according to embodiments of the invention are described in WO2019/046815, WO2015006700, WO2010085699, WO2010099301, WO2010099296, WO2006122442, WO2001081565, and WO1998040510, the contents of which are incorporated herein by reference in their entirety.

Resources for executing the piggyBac method, such as plasmids for expressing transposons/transposases, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a piggyBac system, wherein the at least one gene editor system affects the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population and regulates the expression of at least one checkpoint protein in multiple cells of the second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) The second TIL population is obtained by stimulating it with OKT-3 and culturing it for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivery of at least one gene editor system comprising the piggyBac system, said gene editor system influencing the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population. In some embodiments, the at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

In some embodiments, methods for genetically modifying TIL populations include using non-viral techniques, such as the piggybac method (e.g., piggybac transposons and transposases or piggybac-like transposons and transposases). In some embodiments, the method includes delivering to the TIL: (a) a nucleic acid or amino acid sequence containing a sequence encoding a transposase; and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an mRNA sequence. The mRNA sequence encoding the transposase can be generated in vitro. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is a DNA sequence. The DNA sequence encoding the transposase can be generated in vitro. The DNA sequence may be a cDNA sequence. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an amino acid sequence. The amino acid sequence encoding the transposase can be generated in vitro. A protein super piggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA. In some embodiments, the transposon is a piggybac transposon or a piggybac-like transposon. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In some embodiments, and particularly those in which the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In some embodiments, the piggyBac transposase comprises the amino acid sequence containing SEQ ID NO:14487 of WO2019046815. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or Super piggyBac ^™ (SPB) transposase. In some embodiments, and particularly those in which the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence. In some embodiments of the methods disclosed herein, the transposase is a piggyBac ^™ (PB) transposase. piggyBac (PB) transposases may contain or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to the following sequences:

In some embodiments of the methods disclosed herein, the transposon is a Sleeping Beauty transposon. In some embodiments of the methods disclosed herein, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase. In some embodiments, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween the following sequences:

In some embodiments, including those in which the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof, identity with the following sequences:

V.Gen 2TIL Manufacturing Process

Figures 1 and 2 depict an exemplary TIL process family called Gen 2 (also known as process 2A) containing some of these features. An implementation of Gen 2 is shown in Figure 2.

As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs to enhance their metabolic activity and thus improve relative health status prior to transplantation into a patient, as well as methods for testing said metabolic health status. As generally outlined herein, TILs are typically obtained from patient samples and manipulated to amplify their number prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below.

In some implementations, TILs can be cryopreserved. After thawing, they can also be restimulated before being infused into the patient to increase their metabolism.

In some embodiments, the first amplification (including a process called pre-REP and the process shown as step B in Figures 1 and 36) is shortened to 3 to 14 days, and the second amplification (including a process called REP and the process shown as step D in Figure 1 or 36) is shortened to 7 to 14 days, as discussed in detail below, in the embodiments, and in the accompanying drawings. In some embodiments, the first amplification (e.g., the amplification described as step B in Figure 1 or 36) is shortened to 11 days, and the second amplification (e.g., the amplification described as step D in Figure 1 or 36) is shortened to 11 days. In some embodiments, the combination of the first and second amplifications (e.g., the amplification described as steps B and D in Figure 1 or 36) is shortened to 22 days, as discussed in detail below, in the embodiments, and in the accompanying drawings.

The "steps" denoted as A, B, C, etc. in the following text refer to Figure 1 or Figure 36 and certain embodiments described herein. The order of the steps in the following text and Figures 1 and 36 is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, repetitions of steps, and/or omissions of steps.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples and then expanded into larger populations for further manipulation as described herein, optionally cryopreservation, restimulation as outlined herein, and optionally evaluation of phenotypic and metabolic parameters as indicators of TIL health status.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multifocal sampling is used. In some embodiments, surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells include multifocal sampling (i.e., obtaining samples from one or more tumor sites and/or locations in a patient, as well as from one or more tumors at the same location or closely adjacent to each other). Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be lung tissue. In some embodiments, the applicable TIL is obtained from non-small cell lung cancer (NSCLC). Solid tumors can be skin tissue. In some embodiments, the applicable TIL is obtained from melanoma.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as HBSS.

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is a 10X working stock solution at a concentration of 100 mg/mL.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is a 10X working stock solution of 10,000 IU/mL.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is a 10X working stock solution at a concentration of 10 mg/mL.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatic tumor digests and tumor fragments obtained by digesting or fragmenting tumor samples obtained from patients.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as shown in Figure 1 or Figure 36), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor is 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor is 2mm × 2mm × 2mm. In some embodiments, the tumor is 3mm × 3mm × 3mm. In some embodiments, the tumor is 4mm × 4mm × 4mm.

In some embodiments, the tumor is removed to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of necrotic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of adipose tissue in each mass.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be used to remove these cells.

In some implementations, the cell suspension collected prior to the first amplification step is referred to as the “primary cell population” or “freshly collected” cell population.

In some embodiments, the cells may optionally be frozen after sample collection and cryopreserved before proceeding to the amplification described in step B (which is described in further detail below and in examples in Figures 1 and 36 and Figure 8).

1. Pleural effusion T cells and TIL

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may originate from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may originate from secondary metastatic cancer cells originating from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural transudate. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments where the disclosed methods utilize pleural fluid, ascites fluid or other cystic fluids containing TILs can be used to perform the same methods to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to further processing steps. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to further processing steps. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of viable TILs. If retained in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in further processing steps of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

B. Step B: First Amplification

In some embodiments, the method of the present invention achieves the acquisition of young TILs, which, compared to older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to the subject/patient), enable increased replication cycles at the time of administration to the subject/patient and thus provide additional therapeutic benefits. Characteristics of young TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32: 415-423; Robbins et al., J. Immunol. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, which are each incorporated herein by reference.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (connection region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 1 or Figure 36. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method referred to as Process 1C, as exemplified in Figures 5 and/or 6. In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased.

Following the dissection or digestion of the tumor fragment as described in step A of, for example, Figure 1 or Figure 36, the resulting cells are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 3 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 7 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for about 11 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells.

In some implementations, TIL amplification may be performed using the initial host TIL amplification steps described below and herein (e.g., the steps described in step B of Figure 1 or Figure 36, which may include a process called pre-REP), followed by a second amplification step described below and herein (step D, including a process called rapid amplification protocol (REP) step), followed by optional cryopreservation, and then a second step D described below and herein (including a process called restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic traits and metabolic parameters as described herein.

In TIL culture, the culture is initiated in a 24-well plate, such as using a Costar 24-well cell culture cluster, with a flat bottom (Corning Incorporated, Corning, NY). Each well is seeded with 2 mL of complete culture medium (CM) containing 1 × ^10⁶ tumor digested cells or one tumor fragment and IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digestion cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plate are incubated in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days.

After preparing the tumor fragment, the resulting cells (i.e., the fragment) are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum (or, in some cases, as outlined herein, in the presence of an APC cell population) and 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ primary TIL cells. In some embodiments, the growth medium contains IL-2 or a variant thereof during the initial expansion. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20-30 × ^10⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains IL-2 at concentrations between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL.

In some embodiments, the first amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the first amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the cell culture medium contains an anti-CD3 agonist antibody, such as OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, OKT-3 is present in the cell culture medium at the start of the first amplification (day 0). In some embodiments, OKT-3 is added to the cell culture medium at any time during the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 1 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 2 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 3 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 4 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 5 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 6 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 7 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 8 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 9 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 10 of the first amplification. In some implementations, OKT-3 is added to the cell culture medium on day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 1 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 3 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 6 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 7 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 8 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 9 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 10 and day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 2 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 3 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 4 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 5 of the first amplification.

In some implementations, OKT-3 is added once or more between day 5 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 6 of the first amplification.

In some implementations, OKT-3 is added once or more between day 6 and day 7 of the first amplification.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digest cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plates are cultured in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days. In some embodiments, CM is CM1 as described in the examples, see Example 1. In some embodiments, the first amplification is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 3-14 days, as discussed in the embodiments and figures. In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 7 to 14 days, as discussed in the embodiments and shown in FIG. 4 and FIG. 5, and includes amplification such as that described in step B of FIG. 1 or FIG. 36. In some embodiments, the first amplification in step B is shortened to 10-14 days. In some embodiments, the first amplification is shortened to 11 days, as discussed in the amplification described in step B of FIG. 1 or FIG. 36.

In some embodiments, the first TIL amplification can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the first TIL amplification can be performed for 1 to 14 days. In some embodiments, the first TIL amplification can be performed for 2 to 14 days. In some embodiments, the first TIL amplification can be performed for 3 to 14 days. In some embodiments, the first TIL amplification can be performed for 4 to 14 days. In some embodiments, the first TIL amplification can be performed for 5 to 14 days. In some embodiments, the first TIL amplification can be performed for 6 to 14 days. In some embodiments, the first TIL amplification can be performed for 7 to 14 days. In some embodiments, the first TIL amplification can be performed for 8 to 14 days. In some embodiments, the first TIL amplification can be performed for 9 to 14 days. In some embodiments, the first TIL amplification can be performed for 10 to 14 days. In some embodiments, the first TIL amplification can be performed for 11 to 14 days. In some embodiments, the first TIL amplification can proceed for 12 to 14 days. In some embodiments, the first TIL amplification can proceed for 13 to 14 days. In some embodiments, the first TIL amplification can proceed for 14 days. In some embodiments, the first TIL amplification can proceed for 1 to 11 days. In some embodiments, the first TIL amplification can proceed for 2 to 11 days. In some embodiments, the first TIL amplification can proceed for 3 to 11 days. In some embodiments, the first TIL amplification can proceed for 4 to 11 days. In some embodiments, the first TIL amplification can proceed for 5 to 11 days. In some embodiments, the first TIL amplification can proceed for 6 to 11 days. In some embodiments, the first TIL amplification can proceed for 7 to 11 days. In some embodiments, the first TIL amplification can proceed for 8 to 11 days. In some embodiments, the first TIL amplification can proceed for 9 to 11 days. In some embodiments, the first TIL amplification can proceed for 10 to 11 days. In some embodiments, the first TIL amplification can proceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the first amplification period. In some embodiments, the first amplification period, including, for example, during step B of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, as discussed in the examples and figures, the first amplification process (including a process called pre-REP; for example, step B according to Figure 1 or Figure 36) is shortened to 3 to 14 days. In some embodiments, the first amplification in step B is shortened to 7 to 14 days. In some embodiments, the first amplification in step B is shortened to 10 to 14 days. In some embodiments, the first amplification is shortened to 11 days.

In some embodiments, the first amplification is performed in a closed-system bioreactor, such as step B according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

1. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Transition from the first amplification to the second amplification

In some cases, the host TIL population obtained from the first amplification, including, for example, the TIL population obtained from step B as shown in Figure 1 or Figure 36, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first amplification (referred to as the second TIL population) can undergo a second amplification (which may include an amplification sometimes referred to as REP) and then be cryopreserved as discussed below. Similarly, where the genetically modified TILs are intended for therapeutic use, the first TIL population (sometimes referred to as the host TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be genetically modified before amplification or after the first amplification and before the second amplification for use in a suitable treatment.

In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is stored until a phenotypic analysis for selection is performed. In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is not stored and proceeds directly to the second amplification. In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 10 days after fragmentation. In some implementations, the transition from the first amplification to the second amplification occurs approximately 7 to 14 days after fragmentation. In other implementations, the transition from the first amplification to the second amplification occurs approximately 14 days after fragmentation.

In some embodiments, the transition from the first amplification to the second amplification occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 1 to 14 days after fragmentation. In some embodiments, the first TIL amplification may occur 2 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 5 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 7 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after fragmentation. In some embodiments, the transition occurs 9 to 14 days after fragmentation. In some embodiments, the transition occurs 10 to 14 days after fragmentation. In some embodiments, the transition occurs 11 to 14 days after fragmentation. In some embodiments, the transition occurs 12 to 14 days after fragmentation. In some embodiments, the transition occurs 13 to 14 days after fragmentation. In some embodiments, the transition occurs 14 days after fragmentation. In some embodiments, the transition occurs 1 to 11 days after fragmentation. In some embodiments, the transition occurs 2 to 11 days after fragmentation. In some embodiments, the transition occurs 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 11 days after fragmentation. In some embodiments, the transition occurs 5 to 11 days after fragmentation. In some embodiments, the transition occurs 6 to 11 days after fragmentation. In some embodiments, the transition occurs 7 to 11 days after fragmentation. In some embodiments, the transition occurs 8 to 11 days after fragmentation. In some embodiments, the transition occurs 9 to 11 days after fragmentation. In some embodiments, the transition occurs 10 to 11 days after fragmentation. In some embodiments, the transition occurs 11 days after fragmentation.

In some embodiments, TILs are not stored after the first amplification and before the rapid second amplification, and the TILs undergo the second amplification directly (e.g., in some embodiments, no storage occurs during the transition from steps B to D as shown in Figure 1 or Figure 36). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from the first amplification (the second TIL population) undergo the second amplification directly without a transition period.

In some embodiments, the transition from the first amplification to the second amplification (e.g., step C according to Figure 1 or Figure 36) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

According to this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from MiltenyiBiotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulation of TIGIT expression and downregulation of one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step also includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Second amplification

In some embodiments, the number of TIL cell populations is expanded after collection and initial host treatment, for example, after steps A and B and a transition referred to as step C (as indicated in Figure 1 or Figure 36). This further expansion is referred to herein as a second expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP); and the process indicated in step D of Figure 1 or Figure 36. The second expansion is typically performed in a breathable container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 1 or Figure 36) may be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL amplification may be performed for about 7 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 8 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 9 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 10 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 11 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 12 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 13 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 14 days.

In some embodiments, the second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the procedures indicated in step D of Figure 1 or Figure 36). For example, TILs can be rapidly amplified using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as about 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (available from BioLegend, San Diego, CA). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation to the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of: urinumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, the second amplification period, including, for example, during step D of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step D of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, during rapid expansion and/or secondary expansion, the ratio of TILs to PBMCs and/or antigen-presenting cells is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:100 and 1:200.

In some implementations, REP and/or secondary amplification are performed in culture flasks, where the host TIL is mixed with 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. The medium is replaced (typically 2/3 replaced with fresh medium via aspiration) until the cells are transferred to a replacement growth chamber. The replacement growth chamber comprises G-REX culture flasks and aeration containers, as discussed more fully below.

In some embodiments, the second amplification (which may include a process referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second amplification is shortened to 11 days.

In some embodiments, the REP and/or second amplification can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or aeration dishes (G-REX culture flasks). In some embodiments, the second amplification (including amplification referred to as rapid amplification) is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs suspended in 150 mL of medium can be added to each T-175 culture flask. The TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The T-175 culture flasks can be incubated at 37°C and 5% _CO₂ . Half of the medium can be replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some implementations, on day 7, cells from two T-175 culture flasks can be combined in a 3L bag, and 300 mL of AIM V with 5% human AB serum and 3000 IU/mL IL-2 can be added to 300 mL of TIL suspension. The cell count in each bag is counted daily or every two days, and fresh culture medium is added to maintain the cell count between 0.5 and 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (which may include an amplification referred to as REP, as well as the amplification mentioned in step D of Figure 1 or Figure 36) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing, New Brighton, MN, USA) with a 100 cm aerated silica bottom. 5 × ^10⁶ or 10 × ^10⁶ TILs can be cultured together with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). The G-REX-100 culture flask can be incubated at 37°C in 5% _CO₂ . On day 5, 250 mL of the supernatant can be removed and transferred to a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. TIL precipitate can be resuspended in 150 mL of fresh culture medium containing 5% human AB serum and 3000 IU/mL IL-2, and added back to the initial G-REX-100 culture flask. When TILs are serially amplified in G-REX-100 culture flasks, on day 7, TILs from each G-REX-100 flask can be resuspended in 300 mL of culture medium present in each flask, and the cell suspension can be aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 can be added to each culture flask. The G-REX-100 culture flasks can be incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 can be added to each G-REX-100 culture flask. Cells can be harvested on day 14 of culture. In some implementations, the process uses different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different number of repetitions.

In some embodiments, the second amplification (including an amplification termed REP) is performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some embodiments, two-thirds of the medium is replaced with fresh medium by aspiration. In some embodiments, the alternative growth chamber comprises a G-REX culture flask and a ventilated container, as discussed more fully below.

In some embodiments, a second amplification (including an amplification referred to as REP) is performed, and the process also includes a step of selecting the TIL for superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability assays may be performed after a second amplification (including amplification known as REP amplification) using standard assays known in the art. For example, a trypan blue exclusion assay may be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample may be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, the second amplification of TILs (including amplification referred to as REP) can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., 2008, Jimmunother., 31, 742-751, and Dudley et al., 2003, Jimmunother., 26, 332-342) or aeration G-REX culture flasks. In some embodiments, the second amplification is performed using culture flasks. In some embodiments, the second amplification is performed using aeration G-REX culture flasks. In some embodiments, the second amplification is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs are suspended in approximately 150 mL of culture medium and added to each T-175 culture flask. TILs were cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs used as "feeder" cells, and the cells were cultured in a 1:1 mixture (50/50 medium) of CM and AIM-V supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. T-175 culture flasks were incubated at 37°C and 5% _CO2 . In some embodiments, half of the medium was replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 culture flasks were pooled in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 was added to 300 mL of TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh culture medium can be added to maintain the cell count between approximately 0.5 and approximately 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (including an amplification referred to as REP) is performed in a 500 mL volumetric flask (G-REX-100, Wilson Wolf) with a 100 ^cm² permeable silica bottom. Approximately 5 × ^10⁶ or 10 × ^10⁶ TILs are incubated with irradiated allogeneic PBMCs at a 1:100 ratio in 400 mL of 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The G-REX-100 flask is incubated at 37°C and 5% _CO₂ . In some embodiments, on day 5, 250 mL of the supernatant is removed and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL precipitate is then resuspended in 150 mL of fresh 50/50 medium with 3000 IU/mL IL-2 and added back to the initial G-REX-100 flask. In the embodiment of continuous TIL amplification in G-REX-100 culture flasks, on day 7, the TIL from each G-REX-100 flask is resuspended in 300 mL of culture medium present in each flask, and the cell suspension is aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 is added to each culture flask. The G-REX-100 culture flasks are incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 is added to each G-REX-100 culture flask. Cells are harvested on day 14 of culture. In some embodiments, the process is performed using different centrifugation speeds (400 g, 300 g, 200 g, for 5 minutes) and different number of repetitions.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some implementations, the second amplification medium (e.g., sometimes referred to as CM2 or second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the second amplification is performed in a closed-system bioreactor, such as step D according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve vertical scaling up of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve lateral scaling out of the culture by: (a) performing rapid or second amplification by culturing TILs in a first small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the TILs transferred from the first small-scale culture is cultured in a second small-scale culture for approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 5 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid or second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid or second amplification is performed for a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid or second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid or second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid or secondary amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid or secondary amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains the same components as the cell culture medium used for rapid or secondary expansion after splitting. In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains different components than the cell culture medium used for rapid or secondary expansion after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid or secondary expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

1. Feeder cells and antigen-presenting cells

In some embodiments, the second amplification procedure described herein (e.g., including the amplification described in step D of Figure 1 or Figure 36, and an amplification referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells placed into the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some implementations, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 50 × ^10⁶ TILs. In still other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 25 × ^10⁶ TILs.

In some embodiments, the second amplification procedure described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs in the second expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796A1, the disclosure of which is incorporated herein by reference.

Step E: Collect TIL

Cells can be collected after the second amplification step. In some embodiments, TILs are collected after one, two, three, four, or more amplification steps, as shown in, for example, Figure 1 or Figure 36. In some embodiments, TILs are collected after two amplification steps, as shown in, for example, Figure 1 or Figure 36.

TILs can be collected by any suitable and aseptic method, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the methods of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, collection, such as step E according to Figure 1 or Figure 36, is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, step E according to Figure 1 or Figure 36 is performed according to the process described herein. In some embodiments, the closed system is introduced under aseptic conditions via a syringe to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is employed.

In some embodiments, TIL is collected according to the methods described in the embodiments. In some embodiments, TIL between day 1 and day 11 is collected using the methods described in the steps mentioned herein, such as the day 11 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 24 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 22 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments.

Step F: Final preparation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 1 or Figure 36 and outlined in detail above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, TILs amplified using the APCs of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using the PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VI. Gen 3TIL Manufacturing Process

Without being limited by any particular theory, it is believed that the initiation of a first expansion to initiate T cell activation, followed by a rapid second expansion to enhance T cell activation, as described in the method of the present invention, allows for the preparation of expanded T cells that retain a "younger" phenotype, and therefore it is anticipated that the expanded T cells of the present invention will exhibit higher cytotoxicity against cancer cells compared to T cells expanded by other methods. In particular, it is believed that initiating T cell activation by exposure to anti-CD3 antibodies (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), as taught in the method of the present invention, and then enhancing it by subsequent exposure to additional anti-CD3 antibodies (e.g., OKT-3), IL-2, and APCs, limits or avoids the maturation of T cells in the culture medium, thereby producing a population of T cells with a less mature phenotype, which are less exhausted by culture expansion and exhibit higher cytotoxicity against cancer cells. In some implementations, the rapid second expansion step is broken down into multiple steps to achieve vertical scaling up of culture scale by: (a) performing rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the T cells from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container and culturing the T cells from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the T cells transferred from the first small-scale culture is cultured in the second small-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs. In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some implementations, after rapid expansion, multiple subpopulations contain a therapeutically effective amount of TIL. In some implementations, after rapid expansion, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some implementations, after rapid expansion, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, rapid amplification is performed over a period of approximately 1 to 5 days before being split into multiple steps. In some implementations, the splitting of rapid amplification occurs approximately day 1, day 2, day 3, day 4, or day 5 after the start of rapid amplification.

In some implementations, the split of rapid amplification occurs approximately day 8, 9, 10, 11, 12, or 13 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of rapid amplification occurs approximately day 10 after the start of the first amplification. In another exemplary implementation, the split of rapid amplification occurs approximately day 11 after the start of the first amplification.

In some implementations, rapid amplification is performed for a period of approximately 4 to 11 days after the split. In some implementations, rapid amplification is performed for a period of approximately 3, 4, 5, 6, 7, 8, 9, 10, or 11 days after the split.

In some embodiments, the cell culture medium used for rapid expansion before splitting contains the same components as the cell culture medium used for rapid expansion after splitting. In some embodiments, the cell culture medium used for rapid expansion before splitting contains different components than the cell culture medium used for rapid expansion after splitting.

In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, the fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

In some implementations, rapid second expansion is performed after the T cell activation achieved by initiating the first expansion begins to decrease, slow down, decline, or subside.

In some cases, rapid second expansion occurs when T cell activation, achieved through initiating the first expansion, has decreased by just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, or 47%. The percentages are 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 100%.

In some implementations, the rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

In some implementations, rapid second expansion occurs when T cell activation achieved by initiating the first expansion has decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46%. The percentages are 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after that.

In some cases, rapid second expansion occurs when T cell activation achieved through initiating first expansion has decreased to at most just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%. %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, the reduction in T cell activation achieved by initiating the first expansion is determined by a decrease in the amount of interferon-γ released by T cells in response to antigen stimulation.

In some implementations, the initial expansion of T cells occurs within a period of at most exactly 7 days or about 8 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or approximately 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, the initial expansion of T cells is carried out over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, rapid secondary expansion of T cells takes place over a period of at most exactly or about 11 days.

In some implementations, rapid secondary expansion of T cells is carried out within a period of at most exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, rapid secondary expansion of T cells is carried out over time periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 11 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 8 days, and the rapid second expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 9 days.

In some implementations, the initial expansion of T cells occurs over an 8-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 9 days.

In some implementations, the initial expansion of T cells occurs over a 7-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, T cells are tumor-infiltrating lymphocytes (TILs).

In some implementations, the T cells are bone marrow infiltrating lymphocytes (MIL).

In some implementations, the T cells are peripheral blood lymphocytes (PBLs).

In some implementations, the T cells are obtained from a donor with cancer.

In some implementations, T cells are TILs obtained from tumors removed from patients with cancer.

In some implementations, the T cells are MILs obtained from the bone marrow of patients with hematological malignancies.

In some implementations, the T cells are peripheral blood mononuclear cells (PBMCs) derived from a donor. In some implementations, the donor has cancer. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the donor has a tumor. In some implementations, the tumor is a liquid tumor. In some implementations, the tumor is a solid tumor. In some implementations, the donor has a hematological malignancy.

In some aspects of this disclosure, immune effector cells (e.g., T cells) can be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL separation). In a preferred aspect, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected via apheresis may be washed to remove plasma fractions and optionally placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations. In one aspect, T cells are separated from peripheral blood lymphocytes by lysing erythrocytes and, for example, by depleting monocytes through PERCOLL gradient centrifugation or by countercurrent centrifugation.

In some embodiments, the T cells are PBLs isolated from whole blood or lymphocyte-rich apheresis products from a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematologic malignancy. In some embodiments, PBL is isolated from whole blood or lymphocyte-rich apheresis products using a positive or negative selection method, i.e., removing PBL using markers of the T-cell phenotype (e.g., CD3+CD45+), or removing non-T-cell phenotype cells while leaving PBL. In other embodiments, PBL is separated by gradient centrifugation. After PBL is isolated from donor tissue, the initial amplification of PBL can be initiated according to the initial amplification step of any method described herein by seeding a suitable number of isolated PBLs (approximately 1 × ^10⁷ PBLs in some embodiments) into an initial amplification culture.

An exemplary TIL process containing some of these features, referred to as process 3 (also referred to herein as Gen 3), is depicted in FIG8 (in particular, for example, FIG8B and/or FIG8C and/or FIG8D) and FIG36, and some advantages of this embodiment of the invention compared to Gen 2 are described in FIG1, FIG2, FIG8, FIG30 and FIG31 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). Implementations of Gen 3 are shown in Figures 1, 8, 30, and 36 (particularly, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). Process 2A or Gen 2 is also described in U.S. Patent Publication No. 2018/0280436, which is incorporated herein by reference in its entirety. The Gen 3 process is also described in International Patent Publication WO 2020/096988.

As discussed and generally summarized herein, TILs are derived from patient samples and manipulated using a TIL amplification process described herein and referred to as Gen 3 to amplify their number before transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below. In some embodiments, TILs may be cryopreserved before or after amplification. After thawing, they may also be restimulated to increase their metabolism before infusion into a patient.

In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the process referred to herein as the Rapid Amplification Protocol (REP) and the process shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 7 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (in particular, e.g., Figure 8B and/or Figure 8C)) takes 1 to 7 days, and the rapid second amplification (including the process referred herein as rapid amplification protocol (REP), and the process shown as step D in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) takes 1 to 10 days, as discussed in detail below and in the embodiments and figures. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 9 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some embodiments, the combination of initiating the first amplification and rapid second amplification (e.g., amplification described in Figure 8 (particularly, e.g., Figure 8B and/or Figure 8C as steps B and D) lasts 14-16 days, as discussed in detail below and in the examples and figures. In particular, it is believed that certain embodiments of the invention include an initiating first amplification step, wherein TILs are activated by exposure to an anti-CD3 antibody (e.g., OKT-3) in the presence of IL-2 or by exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody (e.g., OKT-3). In some embodiments, the TILs activated in the initiating first amplification step as described above are a first TIL population, i.e., a primary cell population.

The “step” designations A, B, C, etc. in the following text are non-limiting examples with reference to Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and certain non-limiting embodiments described herein. The order of steps in the following text and Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, step repetitions, and/or step omissions.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples (“primary TILs”) or from circulating lymphocytes (e.g., peripheral blood lymphocytes, including peripheral blood lymphocytes with TIL-like characteristics), and are then expanded into larger populations for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotype and metabolic parameters as indicators of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be any type of cancer, including (but not limited to) breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including (but not limited to) squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the applicable TIL is obtained from malignant melanoma tumors because reports indicate that these tumors have particularly high levels of TIL.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. TILs are cultured from these fragments using enzymatically digested tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociating enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a certain amount of sterile buffer (e.g., HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is 100 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally, cell suspensions obtained from tumors are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as provided in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), the tumor is physically fragmented. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor fragments are 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor fragments are 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2mm × 2mm × 2mm. In some embodiments, the tumor fragment is 3mm × 3mm × 3mm. In some embodiments, the tumor fragment is 4mm × 4mm × 4mm.

In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue in each fragment. In some embodiments, the tumor fragmentation step is an in vitro or ex vivo method.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, the cell suspension prior to initiating the first amplification step is referred to as the “primary cell population” or “freshly acquired” or “freshly isolated” cell population.

In some embodiments, the cells may optionally be frozen after sample separation (e.g., after obtaining a tumor sample and/or after obtaining a cell suspension from a tumor sample) and cryopreserved before proceeding to the amplification described in step B, which is further described in detail below and illustrated in Figure 8 (particularly, for example, Figure 8B).

1. TIL derived from hollow needles/small biopsies

In some implementations, TILs are initially obtained from patient tumor samples (“primary TILs”) obtained through core needle biopsy or similar procedures and then expanded into a larger population for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters.

In some embodiments, patient tumor samples may be obtained using methods known in the art, typically via mini-biopsy, core needle biopsy, needle biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples may be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples may also be liquid tumors, such as those obtained from hematologic malignancies. In some embodiments, the sample may be derived from multiple small tumor samples or biopsies. In some embodiments, the sample may comprise multiple tumor samples from a single tumor from the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three, or four tumors from the same patient. In some embodiments, the sample may comprise multiple tumor samples from multiple tumors from the same patient. The solid tumor may be lung and/or non-small cell lung cancer (NSCLC).

Generally, cell suspensions obtained from the tumor core or fragments are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

In some implementations, if the tumor is metastatic and the primary lesion has been effectively treated/removed in the past, it may be necessary to remove a metastatic lesion. In some implementations, when available, minimally invasive methods are used to remove skin lesions or lymph nodes in the neck or axillary region. In some implementations, skin lesions are removed or small biopsies are taken. In some implementations, lymph nodes are removed or small biopsies are taken. In some implementations, the tumor is melanoma. In some implementations, the small biopsy of melanoma includes a mole or a portion thereof.

In some embodiments, a small biopsy is a perforated biopsy. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin surrounding a suspected mole. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin and removing a circular piece of skin. In some embodiments, a small biopsy is a perforated biopsy and removes a circular portion of the tumor.

In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed along with a small border of normal-looking skin.

In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy and only the most irregular portion of the mole or growth is collected. In some implementations, the small biopsy is an incisional biopsy, and the incisional biopsy is used when other techniques cannot be performed, such as when the suspected mole is very large.

In some implementations, the small biopsy is a lung biopsy. In some implementations, the small biopsy is obtained via bronchoscopy. Typically, bronchoscopy involves inserting a small instrument through the nose or mouth, down the throat, and into the bronchial passage under the patient's anesthesia, where the instrument is used to remove tissue. In some implementations, a transthoracic biopsy may be used when the tumor or growth cannot be reached via bronchoscopy. Typically, for a transthoracic biopsy, the patient is also under anesthesia and a needle is inserted directly through the skin to the suspected site to remove a small tissue sample. In some implementations, a transthoracic biopsy may require interventional radiology (e.g., using X-ray or CT scan-guided needle). In some implementations, the small biopsy is obtained via puncture biopsy. In some implementations, the small biopsy is obtained via endoscopic ultrasound (e.g., an endoscope with a light and placed in the esophagus via the mouth). In some implementations, the small biopsy is obtained surgically.

In some implementations, the small biopsy is a head and neck biopsy. In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy in which a small piece of tissue is removed from the area of abnormal appearance. In some implementations, if the abnormal area is easily accessible, a sample can be collected without hospitalization. In some implementations, if the tumor is deep inside the mouth or throat, the biopsy may need to be performed under general anesthesia in an operating room. In some implementations, the small biopsy is an excisional biopsy. In some implementations, the small biopsy is an excisional biopsy in which the entire area is removed. In some implementations, the small biopsy is a fine-needle aspiration (FNA). In some implementations, the small biopsy is a fine-needle aspiration (FNA) in which cells are extracted (aspirated) from the tumor or mass using a very fine needle attached to a syringe. In some implementations, the small biopsy is a perforated biopsy. In some implementations, the small biopsy is a perforated biopsy in which a suspicious area is removed using perforated forceps.

In some implementations, the small biopsy is a cervical biopsy. In some implementations, the small biopsy is obtained via colposcopy. Typically, the colposcopy method uses a light-assisted magnifying instrument (coloscope) attached to a binocular magnifying lens, which is then used to examine a small portion of the cervix. In some implementations, the small biopsy is a cervical conization/cone biopsy. In some implementations, a small biopsy is a cervical conization/cone biopsy, in which an outpatient procedure may be required to remove a larger piece of tissue from the cervix. In some implementations, in addition to aiding in diagnosis, a cone biopsy can also be used as initial treatment.

The term "solid tumor" refers to an abnormal mass of tissue that typically does not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The histological structure of a solid tumor includes interdependent tissue compartments, comprising the parenchyma (cancer cells) and supporting stromal cells dispersed within it, providing a supportive microenvironment.

In some embodiments, samples from the tumor are obtained in the form of fine-needle aspiration (FNA), core needle biopsy, or mini-biopsy (including, for example, perforated biopsy). In some embodiments, the sample is first placed in a G-REX-10. In some embodiments, when one or two core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-10. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-100. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-500.

FNA can be obtained from skin tumors, including, for example, melanoma. In some implementations, the FNA is obtained from a skin tumor, such as one from a patient with metastatic melanoma. In some cases, the patient with melanoma has previously undergone surgical treatment.

FNA can be derived from lung tumors, including, for example, NSCLC. In some implementations, the FNA is derived from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the NSCLC patient has previously undergone surgical treatment.

The TILs described herein may be obtained from FNA samples. In some cases, FNA samples are obtained or separated from the patient using fine-gauge needles ranging from 18 to 25 gauge. Fine-gauge needles may be 18, 19, 20, 21, 22, 23, 24, or 25 gauge. In some embodiments, the FNA sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

In some cases, the TILs described herein are obtained from core needle biopsy samples. In some cases, the core needle biopsy sample is obtained or separated from the patient using a surgical or medical needle in the range of 11 to 16 gauge. The needle may be 11, 12, 13, 14, 15, or 16 gauge. In some embodiments, the core needle biopsy sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some implementations, the TIL is not derived from tumor digest. In some implementations, the solid tumor core is not fragmented.

In some embodiments, the tumor digest is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically dissociated again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically dissociated a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether it is incubated at 37°C in 5% _CO2 for another 30 minutes. In some implementations, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, obtaining the first TIL population involves a multifocal sampling method.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as Hank's balance salt solution (HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some implementations, the enzyme stock solution may be varied, thus requiring verification of the concentration of the lyophilized stock solution and corresponding modification of the final amount of enzyme added to the digestion mixture.

In some embodiments, the enzyme mixture comprises approximately 10.2 μl of neutral protease (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

2. Pleural effusion T cells and TILs

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may be derived from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells derived from another organ (e.g., breast, ovary, colon, or prostate). In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural effusion. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments of the pleural fluid in the examples of the present invention, ascites fluid or other cyst fluid containing TILs can be used for the same method to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to the contact step. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to the contact step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of live TILs. If retained in untreated pleural fluid, the number of live TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in the contact step is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in the contact step of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

3. Methods for expanding peripheral blood lymphocytes (PBL) from peripheral blood.

PBL Method 1. In some embodiments of the invention, PBL is amplification using the methods described herein. In some embodiments of the invention, the method includes obtaining a PBMC sample from whole blood. In some embodiments, the method includes enriching T cells by using negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC. In some embodiments, the method includes enriching T cells by using magnetic bead-based negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC.

In some embodiments of the present invention, PBL method 1 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. T cells are isolated using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

PBL Method 2. In some embodiments of the invention, PBL is amplified using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells from the PBMCs are enriched by incubating the PBMCs at 37°C for at least three hours and then separating non-adhesive cells.

In some embodiments of the present invention, PBL method 2 is performed as follows: On day 0, the frozen PMBC sample is thawed, and PBMC cells are seeded at 6 million cells per well in 6-well plates of CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, the non-adherent cells (which are PBL) are removed and their number is counted.

PBL Method 3. In some embodiments of the invention, PBL is amplified using PBL Method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection and T cells are selected using a non-CD19+ fraction of the PBMC sample using negative selection.

In some embodiments of the present invention, PBL method 3 is performed as follows: On day 0, frozen PBMCs derived from peripheral blood are thawed and their number is counted. CD19+ B cells are sorted using a CD19 multi-sorting human kit (Miltenyi Biotec). In the non-CD19+ cell fraction, T cells are purified using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

In some embodiments, PBMCs are isolated from whole blood samples. In some embodiments, PBMC samples are used as starting material for amplifying PBL. In some embodiments, the samples are cryopreserved prior to the amplification process. In other embodiments, fresh samples are used as starting material for amplifying PBL. In some embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human pan-T cell isolation kit and an LS column. In some embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art (e.g., CD19 negative selection).

In some embodiments of the invention, the PBMC sample is incubated for a period of time at a temperature required for effective identification of non-adhesive cells. In some embodiments of the invention, the incubation time is approximately 3 hours. In some embodiments of the invention, the temperature is approximately 37°C. The non-adhesive cells are then amplified using the process described above.

In some embodiments, PBMC samples are from subjects or patients who have optionally pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, tumor samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, PBMC samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or longer. In other embodiments, PBMCs are from patients currently receiving an ITK inhibitor regimen (e.g., ibrutinib).

In some implementations, PBMC samples are derived from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor and who are difficult to treat with a kinase inhibitor or an ITK inhibitor (e.g., ibrutinib).

In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment. In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment and have not been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or longer. In other embodiments, PBMCs are derived from patients who have previously been exposed to ITK inhibitors but have not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In some embodiments of the invention, on day 0, cells are selected for CD19+ and sorted accordingly. In some embodiments of the invention, antibody-bound beads are used for selection. In some embodiments of the invention, purified T cells are isolated from PBMCs on day 0.

In some embodiments of the present invention, for patients who have not been pretreated with ibrutinib or other ITK inhibitors, 10-15 mL of erythrocyte sedimentation rate (ESR) amber layer will produce approximately 5 × ^10⁹ PBMCs, which in turn will produce approximately 5.5 × ^10⁷ PBLs.

In some embodiments of the invention, for patients pretreated with ibrutinib or other ITK inhibitors, the amplification process will generate approximately 20 × ^10⁹ PBLs. In some embodiments of the invention, 40.3 × ^10⁶ PBMCs will generate approximately 4.7 × ^10⁵ PBLs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or obtained from any other method known in the art for obtaining PBMCs.

In some embodiments, the PBL is prepared using the method described in U.S. Patent Application Publication No. US 2020/0347350A1, the disclosure of which is incorporated herein by reference.

4. Methods for expanding bone marrow infiltrating lymphocytes (MILs) derived from bone marrow-derived PBMCs

MIL Method 3. In some embodiments of the invention, the method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+/CD33+/CD20+/CD14+ cells, and non-CD3+/CD33+/CD20+/CD14+ cell fractions are sonicated and a portion of the sonicated cell fractions are added back to the selected cell fractions.

In some embodiments of the present invention, MIL method 3 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. Cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using an S3e cell sorter (Bio-Rad). Cells are sorted into two fractions: immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and AML embryonic cell fraction (non-CD3+CD33+CD20+CD14+).

In some embodiments of the invention, PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow via apheresis, aspiration, biopsy, or other similar methods known in the art. In some embodiments, PBMCs are fresh. In other embodiments, PBMCs are cryopreserved.

In some embodiments of the invention, the MIL is expanded from 10-50 mL of bone marrow aspirate. In some embodiments of the invention, 10 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 20 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 30 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 40 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 50 mL of bone marrow aspirate is obtained from the patient.

In some embodiments of the invention, the number of PBMCs produced from about 10-50 mL of bone marrow aspirate is about 5 × ^10⁷ to about 10 × ^10⁷ PBMCs. In other embodiments, the number of PBMCs produced is about 7 × ^10⁷ PBMCs.

In some embodiments of the invention, approximately 5 × ^10⁷ to approximately 10 × ^10⁷ PBMCs produce approximately 0.5 × ^10⁶ to approximately 1.5 × ^10⁶ MILs. In some embodiments of the invention, approximately 1 × ^10⁶ MILs are produced.

In some embodiments of the invention, 12 × ^10⁶ PBMCs derived from bone marrow aspirate yield approximately 1.4 × ^10⁵ MILs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, bone marrow, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or from any other method known in the art for obtaining PBMCs.

In some embodiments, the MIL is prepared using the method described in U.S. Patent Application Publication No. US2020/0347350A1, the disclosure of which is incorporated herein by reference.

Step B: Initiating the first amplification

In some embodiments, the method of the present invention provides younger TILs that may offer additional therapeutic benefits compared to older TILs (i.e., TILs that have undergone further replication in multiple rounds prior to administration to the subject/patient). Characteristics of younger TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32, 415-423; Robbins et al., J. Immunother. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, each of which is incorporated herein by reference.

Following the segmentation or digestion of tumor fragments and/or tumor fragments as described in step A of, for example, Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) under conditions favorable to TIL growth but unfavorable to tumor and other cell growth. In some embodiments, IL-2, OKT-3, and feeder cells are added together with the tumor digest and/or tumor fragments at the start of culture (e.g., on day 0). In some embodiments, the tumor digest and/or tumor fragments are incubated in containers at a concentration of up to 60 fragments per container and with 6000 IU/mL IL-2. In some embodiments, culturing this primary cell population for several days, typically 1 to 8 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, culturing this primary cell population for several days, typically 1 to 7 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells.

In some implementations, TIL amplification may be performed using the steps described in step B of Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), which may include a process called pre-REP or initiating REP and which begins on day 0 and/or from the start of culture containing feeder cells), followed by a rapid second amplification (step D, including a process called the Rapid Amplification Protocol (REP) step) as described in step D below and here, followed by optional cryopreservation, and then a second step D as described below and here (including a process called the restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some implementations, the tumor fragments are between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.

In some embodiments, there are fewer or equal to 240 tumor fragments. In some embodiments, fewer or equal to 240 tumor fragments are placed in fewer or equal to 4 containers. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, fewer or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each container contains fewer or equal to 500 mL of culture medium. In some embodiments, the culture medium contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the culture medium contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains OKT-3. In some embodiments, the culture medium contains 30 ng/mL OKT-3 per container. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells ^per container.

After preparing the tumor fragment, the resulting cells (i.e., fragments of the primary cell population) are cultured in a medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions favorable to TIL growth but unfavorable to tumor and other cell growth, allowing TIL initiation and accelerated growth from day 0. In some embodiments, the tumor digest and/or tumor fragment are incubated with 6000 IU/mL IL-2, antigen-presenting feeder cells, and OKT-3. This primary cell population is cultured for several days, typically 1 to 8 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initial expansion period contains IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for several days, typically 1 to 7 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initiation of the first expansion comprises IL-2 or a variant thereof, along with antigen-presenting feeder cells and OKT-3. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20-30 ^{× 10⁶} IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × 10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the initial amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some embodiments, the initial expansion cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or about 8000 IU/mL IL-2.

In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial expansion medium contains approximately 200 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium also contains IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 2 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the initial cell culture medium for expansion contains OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, the initial amplification cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 6000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the initiation first expansion medium is referred to as "CM" (an abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In some embodiments, CM is CM1 as described in the examples. In some embodiments, the initiation of the first expansion is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initiation first expansion medium or initial cell culture medium or first cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (also referred to herein as feeder cells).

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific). In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some implementations, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisherScientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the specific culture medium described by reference to International PCT Publication WO/1998/030679, which is incorporated herein by reference, can be used in this invention. In that disclosure, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4+, Br, T, ^{Mn2 +} , P, Si4+, V5 ⁺ , Mo6 ⁺ , ^{Ni2 +} , Rb+, Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

Table 4: Concentration of Non-Trace Element Components

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) can be used in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 4 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 5 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG1 or FIG36 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 7 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 4 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8B and/or FIG8C), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 5 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 7 days, as discussed in the embodiments and figures.

In some embodiments, the initiation of first TIL amplification may be performed 1 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 1 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 7 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some implementations, the initiation of the first TIL amplification can be performed 7 days after fragmentation occurs and/or after the start of the first initiation amplification step.

In some implementations, the initial amplification of the TIL can take place for 1, 2, 3, 4, 5, 6, 7, or 8 days. In some implementations, the first TIL amplification can take place for 1 to 8 days. In some implementations, the first TIL amplification can take place for 1 to 7 days. In some implementations, the first TIL amplification can take place for 2 to 8 days. In some implementations, the first TIL amplification can take place for 2 to 7 days. In some implementations, the first TIL amplification can take place for 3 to 8 days. In some implementations, the first TIL amplification can take place for 3 to 7 days. In some implementations, the first TIL amplification can take place for 4 to 8 days. In some implementations, the first TIL amplification can take place for 4 to 7 days. In some implementations, the first TIL amplification can take place for 5 to 8 days. In some implementations, the first TIL amplification can take place for 5 to 7 days. In some implementations, the first TIL amplification can take place for 6 to 8 days. In some implementations, the first TIL amplification can take place for 6 to 7 days. In some implementations, the first TIL amplification can take place for 7 to 8 days. In some implementations, the first TIL amplification can take 8 days. In some implementations, the first TIL amplification can take 7 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, during the initiation of the first amplification, IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included, for example, during step B of the process described herein, according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step B.

In some embodiments, step B of initiating the first amplification (e.g., according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

1. Feeder cells and antigen-presenting cells

In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added during the initiation of the first amplification. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5-7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 6–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 6-7 of the initiation first amplification period. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 or 8 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 8 of the initiation of the first amplification.

In some embodiments, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8B) and those amplifications referred to as pre-REP or initiation REP) requires feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification and during the initiation of the first amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, 2.5 × ^10⁸ feeder cells are used during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per container during the initiation of the first amplification. In some embodiments, 2.5 × ^{10⁸ feeder cells are used per GREX-10} during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per GREX-100 during the initiation of the first amplification.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 has not increased compared to the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells. In other embodiments, the number of feeder cells required to initiate the first expansion is one-quarter, one-third, five-twelfths, or one-half of the number of feeder cells used for rapid second expansion.

In some embodiments, the culture medium for initiating the first expansion contains IL-2. In some embodiments, the culture medium for initiating the first expansion contains 6000 IU/mL IL-2. In some embodiments, the culture medium for initiating the first expansion contains antigen-presenting feeder cells. In some embodiments, the culture medium for initiating the first expansion contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium for initiating the first expansion contains OKT-3. In some embodiments, the culture medium contains 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells. In some embodiments, the culture medium comprises 500 mL of culture medium and ¹⁵ μg OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium comprises 500 mL of culture medium, 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the initiation of the first expansion procedure described herein requires an excess of feeder cells beyond the TIL during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs to initiate the first expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is also possible to initiate the first expansion of TILs using cytokines in combination with two or more of IL-2, IL-15, and IL-21 as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly beneficial for the generation of lymphocytes, and especially T cells as described therein. See, for example, Table 2.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives may be used in the culture medium during step B, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Initiating the transition from the first amplification to the rapid second amplification

In some cases, a population of primary TILs obtained from an initial first amplification (which may include amplification sometimes referred to as pre-REP), including, for example, a population of TILs obtained from step B as indicated in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), may undergo a rapid second amplification (which may include amplification sometimes referred to as a rapid amplification protocol (REP)) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs are intended for therapeutic use, an amplified TIL population from an initial first amplification or an amplified TIL population from a rapid second amplification may be genetically modified for appropriate treatment before the amplification step or after the initial first amplification and before the rapid second amplification.

In some implementations, the TIL obtained from step B as indicated in the initiation of the first amplification (e.g., Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is stored until the phenotype is determined for selection. In some embodiments, the TIL obtained from the initiation of the first amplification (e.g., step B as indicated in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is not stored and is directly subjected to rapid second amplification. In some embodiments, the TIL obtained from the initiation of the first amplification is not cryopreserved after the initiation of the first amplification and before rapid second amplification. In some embodiments, the transition from the initiation of the first amplification to the second amplification occurs approximately 2, 3, 4, 5, 6, 7, or 8 days after tumor fragmentation occurs and/or after the initiation of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 8 days after fragmentation and/or after the start of the first initiation amplification step.

In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1, 2, 3, 4, 5, 6, 7, or 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 6 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 6 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 8 days after fragmentation and/or the start of the first initiation amplification step.

In some embodiments, TILs are not stored after the initial first amplification and before the rapid second amplification, and the TILs undergo rapid second amplification directly (e.g., in some embodiments, they are not stored during the transition from step B to step D as shown in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from which the first amplification was initiated (the second TIL population) undergo rapid second amplification directly without a transition period.

In some embodiments, the transition from initiating the first amplification to the rapid second amplification (e.g., step C according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a GREX-10 or GREX-100. In some embodiments, the closed-system bioreactor is a single bioreactor. In some embodiments, the transition from initiating the first amplification to the rapid second amplification involves a longitudinal scaling up of the container size. In some embodiments, the initiation of the first amplification is carried out in a smaller container compared to the rapid second amplification. In some implementations, the initial amplification is performed in a GREX-100 and the rapid second amplification is performed in a GREX-500.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

In view of this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from Miltenyi Biotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or Figures 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing process also includes a quiescent step. According to some implementations, the quiescent step involves incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Rapid Second Amplification

In some implementations, the TIL cell population is further expanded in number after the collection and initiation of the first expansion (steps A and B) and the transition referred to as step C, as indicated in Figures 8 (in particular, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). This further amplification, referred to herein as rapid second amplification or rapid amplification, may include an amplification process commonly referred to in the art as a rapid amplification procedure (rapid amplification protocol or REP); and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Rapid second amplification is typically performed in a ventilated container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies. In some embodiments, the TIL is transferred to a larger volume container 1, 2, 3, or 4 days after the initiation of rapid second amplification (i.e., on days 8, 9, 10, or 11 of the overall Gen 3 process).

In some embodiments, the rapid second amplification of TIL (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 9 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 1 day after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 2 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 3 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 4 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 5 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 6 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 8 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 9 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 10 days after the initiation of rapid second amplification.

In some embodiments, the rapid second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the process indicated in step D of FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells, wherein feeder cells are added to a final concentration that is two, 2.4, 2.5, 3, 3.5, or 4 times the feeder cell concentration present in the initiating first amplification. For example, TILs can be rapidly expanded by stimulation with nonspecific T cell receptors in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as approximately 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Laritan, NJ or Miltenyi Biotech, Auburn, California), or UHCT-1 (available from BioLegend, San Diego, California). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation with the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL to 60 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3. In some embodiments, the cell culture medium comprises about 60 ng/mL of OKT-3. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium in the rapid second amplification contains 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 500 mL of culture medium and 6000 IU/mL IL-2, 30 μg of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium contains 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells. In some implementations, the culture medium in the rapid second amplification comprises 500 mL of culture medium per container and 6000 IU/mL IL-2, 30 μg OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, during the second amplification period, including, for example, during step D of the process described herein according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step D.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen-presenting cells in rapid expansion and/or secondary expansion is approximately 1:10, approximately 1:15, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:75, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs in rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some implementations, the ratio of TIL to PBMC in rapid amplification and/or secondary amplification is between 1:100 and 1:200.

In some implementations, the REP and/or rapid second amplification are performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 ng/mL OKT3 anti-CD3 antibody, and 6000 IU/mL IL-2 in 150 mL of culture medium, wherein the feeder cell concentration is at least 1.1-fold (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.8X, 2X, 2.1X, 2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, or 4.0X. The culture medium is replaced (usually by removing 2/3 of the used medium and replacing it with an equal volume of fresh medium) until the cells are transferred to a replacement growth chamber. The replacement growth chamber consists of G-REX culture flasks and aeration containers, as discussed in more detail below.

In some embodiments, the rapid second amplification (which may include a process referred to as the REP process) takes 7 to 9 days, as discussed in the examples and figures. In some embodiments, the second amplification takes 7 days. In some embodiments, the second amplification takes 8 days. In some embodiments, the second amplification takes 9 days.

In some embodiments, the second amplification (which may include an amplification referred to as REP, and the amplification mentioned in step D of Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA) with a 100 cm aerated silica bottom, 5 × ^10⁶ or 10 × ^10⁶ TILs can be taken together with PBMCs in 400 mL of supplemented 5% human AB serum at 3000 IU/mL Cells were cultured in a 50/50 medium containing IL-2 and 30 ng/mL anti-CD3 (OKT3). G-REX-100 culture flasks were incubated at 37°C in 5% _CO2 . On day 5, 250 mL of the supernatant was removed and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL precipitate was resuspended in 150 mL of fresh medium containing 5% human AB serum and 6000 IU/mL IL-2 and added back to the original GREX-100 culture flask. As TILs were continuously amplified in GREX-100 culture flasks, they could be transferred to larger culture flasks, such as GREX-500, on day 10 or 11. Cells could be collected on day 14. Cells could be collected on day 15. Cells could be collected on day 16. In some embodiments, the medium was replaced until the cells were transferred to a replacement growth chamber. In some embodiments, two-thirds of the culture medium is replaced by aspirating the used medium and replacing it with an equal volume of fresh medium. In some embodiments, alternative growth chambers include GREX culture flasks and aerated containers, as discussed more fully below. In some embodiments, the process employs different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different numbers of replicates.

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some implementations, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

In some embodiments, the specific culture media described in International Patent Application Publication No. WO 1998/030679 and U.S. Patent Application Publication No. US2002/0076747A1 (which are incorporated herein by reference) are applicable to the present invention. In the disclosures, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , ^{Cr3 +} , Ge4 ⁺ , Se4+, Br, T, ^Mn2+ , P, ^Si4+ , V5 ⁺ , Mo6 ⁺ , ^{Ni2 +, Rb+} , Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) is suitable for use in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, a rapid second amplification (including an amplification termed REP) is performed, and it further includes a step of selecting TILs with superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability can be determined using standard assays known in the art after a rapid second amplification (including amplification known as REP amplification). For example, a trypan blue exclusion assay can be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample can be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve vertical scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a first mini-culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the first mini-culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the TILs transferred from the first mini-culture is cultured in the second mini-culture for a period of approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 5 days; and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid second amplification is performed over a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid second amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid second amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid second expansion before splitting contains the same components as the cell culture medium used for rapid second expansion after splitting. In some embodiments, the cell culture medium used for rapid second expansion before splitting contains different components than the cell culture medium used for rapid second expansion after splitting.

In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

1. Feeder cells and antigen-presenting cells

In some embodiments, the rapid second amplification procedure described herein (e.g., including amplifications such as those described in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In other embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:10, approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 7.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, rapid second expansion requires twice the number of feeder cells of rapid second expansion. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 5 × ^10⁸ feeder cells. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 7.5 × ^10⁸ feeder cells. In other embodiments, the rapid second expansion requires twice (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X the number of feeder cells required to initiate the first expansion.

In some embodiments, the rapid second amplification procedure described herein requires an excess of feeder cells during the rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs. In some embodiments, PBMCs are added to the rapid second amplification at twice the concentration of PBMCs added to initiate the first amplification.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs during rapid second expansion.

2. Cytokines and other additives

The rapid second amplification method described herein typically uses a culture medium containing high doses of cytokines (particularly IL-2), as is known in the art.

Alternatively, it is possible to rapidly expand TILs using combinations of cytokines, as described in U.S. Patent Application Publication No. US2017/0107490 A1, which discloses the use of combinations of two or more IL-2, IL-15, and IL-21, the contents of which are incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having specific uses in many embodiments. The use of combinations of cytokines is particularly beneficial for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Furthermore, additives, such as peroxisome proliferator-activated receptor γ coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, may be used in the culture medium during step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

Step E: Collect TIL

Cells can be collected after the rapid second amplification step. In some embodiments, TILs are collected after one, two, three, four or more amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps (one initiating a first amplification and one rapid second amplification) provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

TILs can be collected in any suitable and aseptic manner, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the process of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some embodiments, step E according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) is performed according to the process described herein. In some embodiments, the closed system is introduced via a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described herein is employed.

In some embodiments, TILs are collected according to the methods described herein. In some embodiments, TILs between days 14 and 16 are collected using the methods described herein. In some embodiments, TILs are collected on day 14 using the methods described herein. In some embodiments, TILs are collected on day 15 using the methods described herein. In some embodiments, TILs are collected on day 16 using the methods described herein.

Step F: Final formulation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and as outlined above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, the TIL amplified using the methods of this disclosure is administered to the patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. The TIL amplified as disclosed herein can be administered via any suitable route known in the art. In some embodiments, the TIL is administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VII. Other Gen 2, Gen 3 and other TIL manufacturing process implementation plans

A. PBMC feeder cell ratio

In some embodiments, the culture medium used for the amplification methods described herein (see, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) includes an anti-CD3 antibody, such as OKT-3. The combination of anti-CD3 antibody and IL-2 induces T cell activation and cell division in TIL populations. This effect is seen in full-length antibodies as well as Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

In some implementations, the number of PBMC feeder cell layers is calculated as follows:

AT cell volume (diameter 10 μm): V = (4/3) ^πr³ = 523.6 ^μm³

B. The volume of G-REX-100(M) with a height of 40 μm (4 cells): V = (4/3) ^πr³ = 4 × ^{10¹² μm³}

C. Number of cells required to fill column B: 4 × ^10¹² μm³ ^{/ 523.6 μm³ =} 7.6 × ^{10⁸ μm³} × 0.64 = 4.86 × ^10⁸

D. The optimal number of cells to activate in 4D space: 4.86 × 10⁸ ^{/ 24} = 20.25 ^{× 10⁶}

E. Extrapolation to feeder cell and TIL numbers for G-REX-500: TIL: 100 × ^10⁶ and feeder cells: 2.5 × ^10⁹

In this calculation, the approximate number of mononuclear cells required to provide TIL activation using icosahedral geometry in a cylinder with a base of 100 ^cm² is used. The experimental result for T cell threshold activation derived from the calculation is approximately 5 × ^10⁸ , which is closely correlated with NCI experimental data, as described in Jin et al., J. Immunother. 2012, 35, 283-292. In (C), the multiplier (0.64) is the random filling density of the equivalent spheres, calculated by Jaeger and Nagel, Science, 1992, 255, 1523-3. In (D), the divisor 24 is the number of equivalent spheres or “Newton number” in 4-dimensional space that can contact similar objects, as described in Musin, Russ. Math. Surv., 2003, 58, 794-795.

In some embodiments, the number of exogenously supplied antigen-presenting feeder cells during the initiation of the first expansion is approximately half the number of exogenously supplied antigen-presenting feeder cells during the rapid second expansion. In some embodiments, the method includes initiating the first expansion in a cell culture medium containing approximately 50% fewer antigen-presenting cells compared to the cell culture medium used in the rapid second expansion.

In other embodiments, the number of exogenously supplied antigen-presenting feeder cells (APCs) during the rapid second amplification is greater than the number of exogenously supplied APCs during the initiation of the first amplification.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs during the rapid second amplification period to the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, or 2.5:1. 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately ¹ ×10⁸, 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, ^1.5 × ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8×10⁸, 1.9× ^10⁸ , 2 ^{×10⁸ ,} 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3×10⁸, 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^{10⁸ ,} 3.1× ^10⁸ , or 3.2×10⁸ ^. The number of exogenously supplied APCs during the rapid second amplification period is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × 10⁸, 3.8 × ^10⁸ , 3.9 × ^10⁸ , ^{4 × 10⁸ ,} 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^10⁸ , 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , ^4.9 × ^10⁸ , 5 × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^10⁸ , 5.3 × ^10⁸ , 5.4 ^{× 10⁸ .} 5.5× ^10⁸ , 5.6× ^10⁸ , 5.7×10⁸, 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3×10⁸, 6.4× ^10⁸ , ^6.5 × ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , ^7.2 × ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , 7.8× ^10⁸ , 7.9× ^10⁸ 8× ^10⁸ , 8.1× ^10⁸ , 8.2×10⁸, 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8×10⁸, 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , ^9.7 × ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or 1×10⁹ APCs .}

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ^1.5 × 10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ² × 10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is exactly or approximately 5 × ^10⁸ APCs.

In some embodiments, the number of APCs (including, for example, PBMCs) added on day 0 of the first expansion is approximately half the number of PBMCs added on day 7 of the first expansion (e.g., day 7 of the method). In some embodiments, the method includes adding antigen-presenting cells to a first TIL population on day 0 of the first expansion and adding antigen-presenting cells to a second TIL population on day 7, wherein the number of antigen-presenting cells added on day 0 is approximately 50% of the number of antigen-presenting cells added on day 7 of the first expansion (e.g., day 7 of the method).

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is greater than the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification.

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 2 × ^10⁶ APCs/ ^cm² to just or about 3 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density of just or about 2 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} Inoculate culture flasks with APCs/cm² at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4×10⁶ ^, or 4.5× ^{10⁶ .}

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 4.0 × ^10⁶ APCs/ ^cm² to just or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from the range of exactly or approximately 4.0 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenously supplied APCs in the rapid second amplification are at exactly or approximately 2.5 × ^10⁶ APCs/ ^cm² , 2.6 × 10⁶ APCs/ ^cm² , 2.7 × ^10⁶ APCs/ ^cm² , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , 3.2 × ^10⁶ , 3.3 × ^10⁶ , 3.4 × ^10⁶ , 3.5 × ^10⁶ , 3.6 × ^10⁶ , 3.7 × ^10⁶ , 3.8 × ^10⁶ , 3.9 × ^10⁶ , 4 × ^10⁶ , 4.1 × ^10⁶ , 4.2 × ^10⁶ , 4.3 × ^10⁶ , 4.4 × ^10⁶ , or 4.5 × ^{10⁶ .} 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8×10⁶, 4.9× ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4×10⁶, 5.5× ^10⁶ , ^{5.6×10⁶ ,} 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , 6× ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , ^6.3 × ^10⁶ , 6.4×10⁶, 6.5× ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , ^6.8 × ^10⁶ , 6.9× ^10⁶ , 7× ^10⁶ Inoculate the culture flasks at densities of 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4× ^10⁶ , or 7.5× ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} The APCs were seeded in culture flasks at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , or 4.5× ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for rapid second amplification were at exactly or approximately 2.5× ^10⁶ APCs/ ^cm² , 2.6× ^10⁶ APCs/ ^cm² , 2.7× ^10⁶ APCs/ ^cm² , 2.8× ^10⁶ , 2.9× ^10⁶ , 3× ^10⁶ , and 3.1×10⁶ ^APCs/cm². 3.2× ^10⁶ , 3.3× ^10⁶ , 3.4×10⁶, 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4×10⁶, 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , 4.5× ^10⁶ , 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8× ^10⁶ , ^4.9 × ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4× ^10⁶ , 5.5× ^10⁶ , ^5.6 × ^10⁶ APCs/cm² were inoculated into culture flasks at densities of 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , ⁶ × ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , 6.3×10⁶, 6.4× ^10⁶ , ^6.5 × ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , 6.8× ^10⁶ , 6.9×10⁶, ⁷ × ^10⁶ , 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4×10⁶, or 7.5× ^{10⁶ .}

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density selected ^from exactly or about 2.5 × ^10⁶ APCs/cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in culture flasks at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in culture flasks at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 2 × ^10⁶ APCs/ ^cm² to exactly or about 3 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied at the rapid second amplification are seeded in the culture flask at a density selected from exactly or about 4 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in the culture flask at a density of exactly or approximately 2 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density of exactly or approximately 4 × ^10⁶ APCs/ ^cm² .

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other implementations, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, etc. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1× ^10⁸ , 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4× ^10⁸ , 1.5× ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9× ^10⁸ , 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , or 3.1×10⁸ ^. The exogenous supply of APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × ^10⁸ , 3.8 × ^10⁸ , 3.9 × ^10⁸ , 4 × ^10⁸ , 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^{10⁸ ,} 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , 4.9 × ^10⁸ , ⁵ × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^{10⁸ . 8} , 5.3× ^10⁸ , 5.4× ^10⁸ , 5.5×10⁸, 5.6× ^10⁸ , 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , 7.2× ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^{10⁸ 8} , 7.8× ^10⁸ , 7.9×10⁸, ⁸ ×10⁸, 8.1× ^10⁸ , 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , 9.7× ^10⁸ , ^9.8 × ^10⁸ , 9.9× ^10⁸ , or 1× ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 1 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 1 × ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 7.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 2 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 4.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 5.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or about 5 × ^10⁸ APCs (including, for example, PBMCs).

In some embodiments, the number of APC (including, for example, PBMC) layers added on day 0 of the first amplification is approximately half the number of APC (including, for example, PBMC) layers added on day 7 of the rapid second amplification. In some embodiments, the method includes adding antigen-presenting cell layers to a first TIL population on day 0 of the first amplification and adding antigen-presenting cell layers to a second TIL population on day 7, wherein the number of antigen-presenting cell layers added on day 0 is approximately 50% of the number of antigen-presenting cell layers added on day 7.

In other implementations, the number of exogenously supplied APC (including, for example, PBMC) layers on day 7 of the rapid second amplification is greater than the number of exogenously supplied APC (including, for example, PBMC) layers on day 0 of the initiation of the first amplification.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about three cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1.5 to just or about 2.5 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about two cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers, and day 7 of the rapid second amplification occurs with an average thickness of just or approximately 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3. It occurs in the presence of layered APCs (including, for example, PBMCs) of 8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1 cell layer to just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 to just or about 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1, 2, or 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9, or 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is exactly or approximately 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from... Exactly or approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1 :4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.0 × ^10⁶ APCs/ ^cm² to about 4.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 2.5 × ^10⁶ APCs/ ^cm² to about 7.5 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.5 × ^10⁶ APCs/ ^cm² to about 3.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 3.5 × ^10⁶ APCs/ ^cm² to about 6.0 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 2.0 × ^10⁶ APCs/ ^cm² to about 3.0 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 4.0 × ^10⁶ APCs/ ^cm² to about 5.5 × ^10⁶ APCs/ ^cm² .

A. Optional cell culture medium components

1. Anti-CD3 antibody

In some embodiments, the culture medium used for the amplification methods described herein (including an amplification method known as REP, see, for example, Figures 1 and 8 (especially, for example, Figure 8B)) includes an anti-CD3 antibody. The combination of the anti-CD3 antibody and IL-2 induces T cell activation and cell division in a TIL population. This effect can be seen with both full-length antibodies and Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California). See Table 1.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California).

2,4-1BB (CD137) agonist

In some embodiments, the cell culture medium initiating the first amplification and/or rapid second amplification contains a TNFRSF agonist. In some embodiments, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule may contain any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The 4-1BB agonist or 4-1BB binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies); monoclonal antibodies (including full-length monoclonal antibodies); polyclonal antibodies; multispecific antibodies (e.g., bispecific antibodies); human, humanized, or chimeric antibodies; and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to 4-1BB, such as scFv molecules. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the 4-1BB agonist used in the methods and compositions disclosed in this invention includes anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectin, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. Agonistant anti-4-1BB antibodies are known to induce strong immune responses. (Lee et al., PLOSOne 2013, 8, e69677). In some embodiments, the 4-1BB agonist is an agonistant anti-4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In some embodiments, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utumumab, or urilumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

In some embodiments, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In some embodiments, polymeric 4-1BB agonists, such as trimeric or hexameric 4-1BB agonists (having three or six ligand-binding domains), can induce the formation of desirable receptor (4-1BBL) clusters and internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins containing three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic 4-1BB antibodies and fusion proteins induce strong immune responses. In some embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO:40) with high affinity and agonistic activity. In some embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO:40). In some embodiments, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO:41). The amino acid sequences of the 4-1BB antigens bound by the 4-1BB agonist or binding molecule are summarized in Table 5.

Table 5.4-1 shows the amino acid sequence of the BB antigen.

In some embodiments, the described compositions, processes, and methods include a 4-1BB agonist that binds to human or rodent 4-1BB at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at _{a KD} of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include 4-1BB agonists that _bind to human or rodent 4-1BB at a k-association rate of about 7.5 × ^{10⁵ 1} /M·s or faster, at a k _-association rate of about 7.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ 1/M·s or faster, at a k- _association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include binding to human or rodent 4-1BB at a k _{-dissociation} rate of about 2 × ^10⁻⁵ 1/s or _slower , at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about _2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 × ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/s or _slower , at about 2.6 × ^10⁻⁵ 1/s or _slower , or at about 2.7 × ^10⁻⁵ 1/s or slower. 4-1BB agonists that bind to human or rodent 4-1BB at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include a _4-1BB agonist that binds to human or rodent 4-1BB at an _IC50 of about ₁₀ nM or less, at about 9 nM or less, at about ₈ nM or less, at about 7 nM or less, at about ₆ nM or less, at about ₅ nM or less, at about ₄ nM or less, at about 3 nM or less, at about ₂ nM or less, or at about 1 nM or less _{. 50} combines with 4-1BB in humans or rodents.

In some embodiments, the 4-1BB agonist is utumumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant, or biosimilar thereof. Utumumab is available from Pfizer. Utumumab is an immunoglobulin G2-λ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of utumumab is illustrated in Table 6. Utumumab contains glycosylation sites at Asn59 and Asn292; intrachain disulfide bridges in the heavy chain at positions 22-96 ( _VH - _VL ), 143-199 ( _CH1 - _CL ), 256-316 ( _CH2 ), and 362-420 ( _CH3 ); and intrachain disulfide bridges at positions 22'-87' ( _VH - _VL ) and 136'-195' ( _CH1 -CL _). Intrachain disulfide bridges of the light chain; interchain heavy chain-heavy chain disulfide bridges located at IgG2A isoform positions 218-218, 219-219, 222-222 and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222 and 225-225 and at IgG2B isoform positions 219-130(2), 222-222 and 225-225; and interchain heavy chain-light chain disulfide bridges located at IgG2A isoform positions 130-213'(2), IgG2A/B isoform positions 218-213' and 130-213' and at IgG2B isoform positions 218-213'(2). The preparation and characterization of utumumab, as well as its variants and fragments, are described in U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678 and International Patent Application Publication No. WO 2012/032433 A1, the disclosures of which are incorporated herein by reference. The preclinical characteristics of utumumab are described in Fisher et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utumumab in various hematologic and solid tumor indications include US National Institutes of Health (NIH) clinicaltrials.gov identification numbers NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:42 and the light chain provided in SEQ ID NO:43. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utumumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:44, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:45, along with its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of utumumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity, such as 97%, 98%, 99%, or 100%, with an amino acid sequence that is 97%, 98%, 99%, or 100% sequence identity with a reference drug or reference biological product, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, which is utumumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, which is utumumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab.

Table 6. Amino acid sequences of 4-1BB agonist antibodies associated with utumumab.

In some embodiments, the 4-1BB agonist is the monoclonal antibody urogenumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biosimilar thereof. Ureogenumab is available from Bristol-Myers Squibb and Creative Biolabs. Ureogenumab is an immunoglobulin G4-κ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of urogenumab is illustrated in Table 7. Urelimab contains N-glycosylation sites at positions 298 (and 298”); intrachain disulfide bridges in the heavy chain at positions 22–95 ( _VH – _VL ), 148–204 ( _CH1 – _CL ), 262–322 ( _CH2 ), and 368–426 ( _CH3 ) (and at positions 22”–95”, 148”–204”, 262”–322”, and 368”–426”); and intrachain disulfide bridges in the heavy chain at positions 23”–88” ( _VH – _VL ) and 136”–196” ( _CH1 –CL _). (and intra-chain disulfide bridges at positions 23”'-88”' and 136”'-196”'); inter-chain heavy chain-heavy chain disulfide bridges at positions 227-227” and 230-230”; and inter-chain heavy chain-light chain disulfide bridges at positions 135-216”' and 135”-216”'. The preparation and characterization of urogenumab, as well as its variants and fragments, are described in U.S. Patent Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical characteristics of urogenumab are described in Segal et al., Clin. Cancer. Res. 2016, available at http://dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical trials of ureglucumab in various hematologic and solid tumor indications include NCT01775631, NCT02110082, NCT02253992, and NCT01471210 from the National Institutes of Health (NIH) clinicaltrials.gov.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:52 and the light chain provided in SEQ ID NO:53. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urinumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:54, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:55, and its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of urogenumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is uroreglumab.

Table 7. Amino acid sequences of 4-1BB agonist antibodies associated with urilumab.

In some embodiments, the 4-1BB agonist is selected from the group consisting of: 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), produced by a cell line registered as ATCC No. HB-11248 and disclosed in U.S. Patent No. 6,974,863. Antibodies disclosed in the following countries include: 5F4 (BioLegend 31 1503), C65-485 (BDPharmingen 559446), antibodies disclosed in US Patent Application Publication No. 2005/0095244, antibodies disclosed in US Patent No. 7,288,638 (e.g., 20H4.9-IgG1 (BMS-663031)), antibodies disclosed in US Patent No. 6,887,673 (e.g., 4E9 or BMS-554271), and antibodies disclosed in US Patent No. 7,214,493. Antibodies disclosed in U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, U.S. Patent No. 6,905,685 (e.g., 4E9 or BMS-554271), U.S. Patent No. 6,362,325 (e.g., 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), U.S. Patent No. 6,974,863 (e.g., 53A2), and U.S. Patent No. 6,210,669 (e.g., 5E9 or 5E2). The antibodies described in U.S. Patent No. 5,928,893 (1D8, 3B8, or 3E1), U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, and International Patent Application Publications Nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, as well as fragments, derivatives, conjugates, variants, or bioanalytes thereof, wherein the disclosure of each of the foregoing patents or patent applications is incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein described in the following: International Patent Application Publications No. WO 2008/025516 A1, WO 2009/007120A1, WO 2010/003766 A1, WO 2010/010051 A1 and WO 2010/078966 A1; U.S. Patent Application Publication No. US 201 The disclosures of U.S. Patent Nos. 1/0027218A1, 2015/0126709 A1, 2011/0111494A1, 2015/0110734 A1, and 2015/0126710 A1; and U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, are incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic fusion protein as depicted in structure IA (C-terminal Fc antibody fragment fusion protein) or structure IB (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof (see Figure 18). In structures IA and IB, the cylinder refers to an individual polypeptide-binding domain. Structures IA and IB contain three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL (4-1BB ligand, CD137 ligand (CD137L), or tumor necrosis factor superfamily member 9 (TNFSF9)) or antibodies binding to 4-1BB. These TNFRSF binding domains fold to form a trivalent protein, which is then linked to a second trivalent protein via an IgG1-Fc (including _CH3 and _CH2 domains). The IgG1-Fc then serves to link the two trivalent proteins together via disulfide bonds (elongated ellipses), thereby stabilizing the structure and providing an agonist capable of linking the intracellular signaling domains of six receptors to signaling proteins to form a signaling complex. A TNFRSF binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys. Any scFv domain design can be used, such as those described in: de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad et al., Clin. & Dev. Immunol. 2012, 980250; Monnier et al., Antibodies, 2013, 2, 193-208; or other references incorporated herein by reference. The structure of this form of fusion protein is described in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of structures I-A provided in Figure 18 are shown in Table 8. The Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO: 62), a complete hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). Preferred adapters for linking C-terminal Fc antibodies are selected from the embodiments provided in SEQ ID NO: 63 to SEQ ID NO: 72, including adapters suitable for fusing other polypeptides.

Table 8. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with C-terminal Fc-antibody fragment fusion protein design (structure I-A).

The amino acid sequences of the other polypeptide domains of structures I-B provided in Figure 18 are shown in Table 9. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SED ID NO:74 to SEQ ID NO:76.

Table 9. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with N-terminal Fc-antibody fragment fusion protein design (structures I-B).

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B includes one or more 4-1BB binding domains selected from the group consisting of: variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains selected from the variable heavy and variable light chains described in Table 10, any combination of the aforementioned variable heavy and variable light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:77. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a soluble 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:78.

In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:43 and SEQ ID NO:44, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the _VH and _VL sequences provided in Table 10, wherein the _VH and _VL domains are connected by linkers.

Table 10. Applicable to the 4-1BB binding domain in fusion proteins or other polypeptide domains applicable to scFv 4-1BB agonist antibodies.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stem region (which promotes trimerization and provides a distance from the cell membrane, but is not part of the 4-1BB binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some embodiments, the 4-1BB agonist is a BPS Bioscience 4-1BB agonist antibody, catalog number 79097-2, available from BPS Bioscience in San Diego, California, USA. In other embodiments, the 4-1BB agonist is a Creative Biolabs 4-1BB agonist antibody, catalog number MOM-18179, available from Creative Biolabs in Shirley, NY, USA.

3. OX40 (CD134) agonist

In some embodiments, the TNFRSF agonist is an OX40 (CD134) agonist. The OX40 agonist can be any OX40-binding molecule known in the art. The OX40-binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX40. The OX40 agonist or OX40-binding molecule may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The OX40 agonist or OX40-binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized or chimeric antibodies, and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to OX40, such as scFv molecules. In some embodiments, the OX40 agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the OX40 agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the OX40 agonist used in the methods and compositions of this disclosure includes anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 adenettin, anti-OX40 domain antibodies, single-chain anti-OX40 fragments, heavy-chain anti-OX40 fragments, light-chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. In some embodiments, the OX40 agonist is an agonistic anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

In some embodiments, the OX40 agonist or OX40 binding molecule may also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun et al., J. Immunother. 2009, 182, 1481-89. In some embodiments, poly-OX40 agonists, such as trimeric or hexameric OX40 agonists (having three or six ligand-binding domains), can induce clustering of the preferred receptor (OX40L) and the formation of internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic OX40 antibodies and fusion proteins can induce strong immune responses. (Curti et al., Cancer Res. 2013, 73, 7189-98). In some embodiments, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficiently reducing toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC), such as NK cell toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the OX40 agonist is characterized by binding to human OX40 (SEQ ID NO: 85) with high affinity and agonistic activity. In some embodiments, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 85). In some embodiments, the OX40 agonist is a binding molecule that binds to mouse OX40 (SEQ ID NO: 86). Table 11 summarizes the amino acid sequences of the OX40 antigen bound to the OX40 agonist or binding molecule.

Table 11. Amino acid sequence of OX40 antigen.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind human or rodent OX40 at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at a _KD of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that _bind to human or rodent OX40 at a k-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at a k _{-dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about 2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 _× ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/ _s or slower, and at about 2.6 × ^10⁻⁵ 1/s or slower. It binds to human or rodent OX40 at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.7 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at an _IC50 of about 10 nM or less, at an IC50 of about 9 nM or less, at an _IC50 of about 8 nM or less _, at an _IC50 of about 7 nM or less, at an IC50 of about 6 nM or less, at an _IC50 of about ₅ nM or less, at an IC50 of about 4 nM or less, at an _IC50 of about 3 nM or less, at _{an IC50} of about 2 nM or less, or at an _IC50 of about 1 nM or less.

In some implementations, the OX40 agonist is tavoricumab, also known as MEDI 0562 or MEDI-0562. Tavoricumab is available from MedImmune, a subsidiary of AstraZeneca. Tavoricumab is a humanized and chimeric monoclonal antibody against immunoglobulin G1-κ [Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)]. The amino acid sequence of tavoricumab is illustrated in Table 12. Tavocitumab contains N-glycosylation sites at positions 301 and 301", with fucosylated complex biantennary CHO-type glycans; intrachain disulfide bridges in the heavy chain at positions 22-95 ( _VH - _VL ), 148-204 ( _CH1 - _CL ), 265-325 ( _CH2 ), and 371-429 ( _CH3 ) (and at positions 22"-95", 148"-204", 265"-325" and 371"-429"); and intrachain disulfide bridges in the heavy chain at positions 23"-88" ( _VH - _VL ) and 134"-194" ( _CH1 -CL ₎ . Intra-chain disulfide bridges of the light chain at positions 23”'-88”' and 134”'-194”'; inter-chain heavy chain-heavy chain disulfide bridges at positions 230-230” and 233-233”'; and inter-chain heavy chain-light chain disulfide bridges at positions 224-214”' and 224”-214”'. Current clinical trials of tavoricumab in various solid tumor indications include NCT02318394 and NCT02705482 from the National Institutes of Health clinicaltrials.gov.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:87 and the light chain provided in SEQ ID NO:88. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of tavoricumab. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:89, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:90, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90 _, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises an scFv antibody, said scFv antibody comprising _VH and _VL regions _, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of the OX40 agonist approved by a regulatory agency, referencing tavoricumab. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab.

Table 12. Amino acid sequences of OX40 agonist antibodies associated with tavoricumab.

In some embodiments, the OX40 agonist is 11D4, a fully human antibody available from Pfizer. The preparation and characterization of 11D4 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is set forth in Table 13.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:97 and the light chain provided in SEQ ID NO:98. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:99, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:100, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100 _, respectively. In some embodiments, the OX40 agonist comprises _VH and VL _regions , each at least 97% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody approved by a regulatory agency with reference to 11D4. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug product or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 11D4.

Table 13. Amino acid sequences of OX40 agonist antibodies associated with 11D4.

In some embodiments, the OX40 agonist is 18D8, a fully human antibody available from Pfizer. The preparation and characterization of 18D8 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is set forth in Table 14.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:107 and the light chain provided in SEQ ID NO:108. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively.

In some embodiments, the OX40 agonist comprises a heavy chain and a light chain CDR or variable region (VR) of 18D8. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:109, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:110, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO: ₁₁₀ , respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to 18D8. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence having at least 97%, such as 97%, 98%, 99%, or 100%, of a reference drug product or reference biological product, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 18D8.

Table 14. Amino acid sequences of OX40 agonist antibodies associated with 18D8.

In some embodiments, the OX40 agonist is Hu119-122, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu119-122 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is set forth in Table 15.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:117, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:118 and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu119-122. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122.

Table 15. Amino acid sequences of OX40 agonist antibodies associated with Hu119-122.

In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu106-222 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is set forth in Table 16.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:125, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:126, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 96% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu106-222. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222.

Table 16. Amino acid sequences of OX40 agonist antibodies associated with Hu106-222.

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B12). MEDI6469 is a murine monoclonal antibody. (Weinberg et al., J. Immunother. 2006, 29, 575-585). In some embodiments, the OX40 agonist is an antibody produced from a 9B12 hybridoma and hosted by Biovest Corporation (Malvern, MA, USA), as described in Weinberg et al., J. Immunother. 2006, 29, 575-585, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the antibody comprises the CDR sequence of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of MEDI6469.

In some embodiments, the OX40 agonist is L106 BD (Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the CDR of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the CDR of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some implementations, the OX40 agonist is a murine monoclonal antibody anti-mCD134/mOX40 (clone OX86), available from BioXcell's InVivoMAb in West Lebanon, New Hampshire.

In some embodiments, the OX40 agonist is selected from the OX40 agonists described below: International Patent Application Publications WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191 and WO 2014/148895; European Patent Application EP 0672141; US Patent Application Publication US2010/136030, ... US Nos. 2014/377284, 2015/190506, and 2015/132288 (including clones 20E5 and 12H3); and US U.S. Patents 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the OX40 agonist is an OX40 agonistic fusion protein as depicted in structures I-A (C-terminal Fc-antibody fragment fusion protein) or I-B (N-terminal Fc-antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof. The characteristics of structures I-A and I-B have been described above and in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequences of the polypeptide domains of structure I-A provided in Figure 18 are shown in Table 9. The Fc domain preferably comprises an intact constant domain (amino acids 17-230 of SEQ ID NO: 62), an intact hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). The preferred adapter for linking the C-terminal Fc antibody can be selected from the embodiments provided in SEQ ID NO:63 to SEQ ID NO:72, including adapters suitable for fusing other peptides. Similarly, the amino acid sequences of the polypeptide domains of structures I-B provided in Figure 18 are shown in Table 10. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SEQ ID NO:74 to SEQ ID NO:76.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40 binding domains selected from the group consisting of: the variable heavy and light chains of tavocisumab, the variable heavy and light chains of 11D4, the variable heavy and light chains of 18D8, the variable heavy and light chains of Hu119-122, the variable heavy and light chains of Hu106-222, the variable heavy and light chains selected from the variable heavy and light chains described in Table 17, any combination of the aforementioned variable heavy and light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 133. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the soluble OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 134. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 135.

In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:127 and SEQ ID NO:128, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO: 125 and SEQ ID NO: 126, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and VL regions, respectively, that are at least 95% identical to the _VH and _VL sequences provided in Table 17, wherein the _VH and _{VL regions} are connected by linkers.

Table 17. OX40 binding domains suitable for use as fusion proteins (e.g., structures I-A and I-B) or other polypeptide domains suitable for use as scFv OX40 agonist antibodies.

In some embodiments, the OX40 agonist is an OX40-agonistic single-chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain further comprising an additional domain at an N-terminus and/or a C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble OX40-binding domain; (ii) a first peptide linker; (iii) a second soluble OX40-binding domain; (iv) a second peptide linker; and (v) a third soluble OX40-binding domain further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domains are Fab or Fc fragment domains, wherein each of the soluble OX40-binding domains lacks a stem region (which facilitates trimerization and provides a distance from the cell membrane, but is not part of the OX40-binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain; (ii) a first peptide linker; (iii) a second soluble TNF superfamily cytokine domain; (iv) a second peptide linker; and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily cytokine domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonist fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In some embodiments, the OX40 agonist is an OX40-agonistic scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some implementations, the OX40 agonist is Creative Biolabs' OX40 agonist monoclonal antibody MOM-18455, which is available from Creative Biolabs in Sherry, New York, USA.

In some implementations, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, which is available from BioLegend, Inc., San Diego, California, USA.

B. Optional cell viability analysis

Optionally, after initiating the first expansion (sometimes referred to as initial bulk expansion), cell viability can be determined using standard assays known in the art. Therefore, in some embodiments, the method includes performing cell viability determination after initiating the first expansion. For example, a trypan blue exclusion assay can be performed on the bulk TIL sample, which selectively labels dead cells and allows for viability assessment. Other assays used to test viability may include (but are not limited to) an alamar blue assay and an MTT assay.

1. Cell counting, viability, and flow cytometry

In some embodiments, cell count and/or viability are measured. Expression of biomarkers (e.g., but not limited to CD3, CD4, CD8, and CD56, and any other biomarkers disclosed or described herein) can be measured by flow cytometry using a FACSCanto ^™ flow cytometer (BD Biosciences), with antibodies, such as (but not limited to) those available from BD Biosciences (BD Biosciences, San Jose, CA). Cells can be manually counted using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art (including, but not limited to, trypan blue staining). Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0282694, which is incorporated herein by reference in its entirety. Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0280436 or International Patent Application Publication No. WO/2018/081473, both of which are incorporated herein by reference in their entirety for all purposes.

In some cases, the primary TIL population can be immediately cryopreserved using the methods discussed below. Alternatively, the primary TIL population can undergo re-expansion (REP) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs will be used in therapy, the primary or REP TIL population can be genetically modified for appropriate treatment.

2. Cell Culture

In some embodiments, the method for amplifying TILs (including those discussed above and illustrated in Figures 1 and 8, particularly those exemplified in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may include the use of about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the culture medium is serum-free. In some embodiments, the culture medium used to initiate the first amplification is serum-free. In some embodiments, the culture medium used to initiate the second amplification is serum-free. In some embodiments, the culture medium used to initiate both the first and second amplifications (also referred to as rapid second amplification) is serum-free. In some embodiments, TIL number amplification uses no more than one type of cell culture medium. Any suitable cell culture medium can be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, CA). In this regard, the method of the present invention advantageously reduces the amount and number of culture media types required for TIL number amplification. In some embodiments, TIL number amplification may include feeding cells at a frequency not exceeding once every three or four days. Amplifying cell numbers in an aerated container simplifies the procedure required for cell number amplification by reducing the feeding frequency needed.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME)-free.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for a period of about 1 to 8 days, for example, about 7 days as the initiation of the first amplification, or about 8 days as the initiation of the first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for a period of about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for about 1 to 7 days (e.g., about 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TILs to a second ventilated container and amplifying the number of TILs in the second ventilated container for about 7 to 14 days or about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days, about 10 days, or about 11 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for approximately 1 to 7 days (e.g., approximately 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium comprising IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for approximately 7 to 11 days (e.g., approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, or approximately 11 days), the second ventilated container containing a cell culture medium comprising IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, TILs are amplified in a breathable container. Breathable containers have been used to amplify TILs, using PBMCs, and employing methods, compositions, and apparatus known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In some embodiments, TILs are amplified in a breathable bag. In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the Xuri Cell Expansion System W25 (GE Healthcare)). In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the WAVE bioreactor system, also known as the Xuri Cell Expansion System W5 (GE Healthcare)). In some embodiments, the cell expansion system includes a breathable cell bag with a volume selected from the group consisting of: about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

In some embodiments, TILs can be expanded in G-REX culture flasks (available from Wilson Wolf Manufacturing). Such embodiments expand the cell population from approximately 5 × ^10⁵ cells/cm² to 10 × ^10⁶ to 30 × ^10⁶ cells/cm². In some embodiments, this is not performed feedering. In some embodiments, this is not performed feedering, as long as the culture medium in the G-REX culture flask is at a height of approximately 10 cm. In some embodiments, this is not performed feedering, but one or more cytokines are added. In some embodiments, the cytokines are added as a bolus, without needing to be mixed with the culture medium. Such containers, apparatuses, and methods are known in the art and have been used to amplify TILs, and include those described in the following: U.S. Patent Application Publication No. US2014/0377739A1, International Publication No. WO 2014/210036A1, U.S. Patent Application Publication No. US 2013/0115617A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Patent No. US 8,809,050 B2, International Publication No. WO 2011/072088A2, U.S. Patent Application Publication No. US2016/0208216A1, U.S. Patent Application Publication No. US2012/0244133 A1, International Publication No. WO 2012/129201A1, and U.S. Patent Application Publication No. US2013/0102075. The disclosures of U.S. Patent No. A1, U.S. Patent No. 8,956,860 B2, International Publication No. WO 2013/173835A1, and U.S. Patent Application Publication No. US2015/0175966 A1 are incorporated herein by reference. Similar processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

Optional gene knockdown or gene knockout in C.TIL

In some embodiments, the amplified TILs of the present invention are further manipulated before, during, or after the amplification step, including during a closed, aseptic manufacturing process (each as provided herein), to temporarily alter protein expression. In some embodiments, the temporarily altered protein expression is due to temporary gene editing. In some embodiments, the amplified TILs of the present invention are treated with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in TILs. In some embodiments, TFs and/or other molecules capable of temporarily altering protein expression provide altered tumor antigen expression and/or altered number of tumor antigen-specific T cells in the TIL population.

In some embodiments, the method includes gene editing of a TIL population. In some embodiments, the method includes gene editing of a first TIL population, a second TIL population, and/or a third TIL population.

In some embodiments, the invention includes gene editing via nucleotide insertion, such as via ribonucleic acid (RNA) insertion, including the insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA) into a TIL population to promote or inhibit the expression of one or more proteins, as well as a combination of promoting one group of proteins and inhibiting another group of proteins.

In some embodiments, the amplified TILs of the present invention undergo a temporary alteration of protein expression. In some embodiments, the temporary alteration of protein expression occurs in the host TIL population prior to the first amplification, including, for example, the TIL population obtained from step A as indicated in, for example, Figure 8 (particularly Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs during the first amplification, including, for example, the TIL population obtained from step B as indicated in Figure 8 (e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs after the first amplification, including, for example, the TIL population transitioning between the first and second amplifications (e.g., the second TIL population described herein), the TIL population obtained from, for example, step B as indicated in Figure 8, and included in step C. In some embodiments, the temporary alteration of protein expression occurs in the main TIL population prior to the second amplification, including, for example, the TIL population obtained from, for example, step C as indicated in Figure 8, and prior to its amplification in step D. In some embodiments, the temporary alteration of protein expression occurs during a second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8 (e.g., a third TIL population). In some embodiments, the temporary alteration of protein expression occurs after the second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8.

In some embodiments, the method for temporarily altering protein expression in a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. In some embodiments, the method for temporarily altering protein expression in a TIL population includes a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include transfection steps using methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

In some embodiments, temporary alteration of protein expression induces an increase in stem memory T cells (TSCMs). TSCMs are early progenitor cells of antigen-experiencing central memory T cells. TSCMs typically exhibit the long-term survival, self-renewal, and pluripotent capabilities that define stem cells and are generally required for the production of effective TIL products. In mouse models of adoptive cell transfer, enhanced antitumor activity of TSCMs compared to other subsets of T cells has been demonstrated. In some embodiments, temporary alteration of protein expression induces a TIL population with a composition containing a high proportion of TSCMs. In some embodiments, temporary alteration of protein expression induces an increase in the percentage of TSCMs of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, a temporary alteration of protein expression causes a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCM in a TIL population. In some embodiments, a temporary alteration of protein expression causes a TIL population with at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM. In some implementations, a temporary alteration in protein expression results in a therapeutic TIL population having at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM.

In some implementations, a temporary alteration of protein expression induces antigen-experiencing T-cell rejuvenation. In some implementations, rejuvenation includes, for example, increased proliferation, increased T-cell activation, and/or increased antigen recognition.

In some implementations, temporarily altering protein expression changes the expression of a large portion of T cells to preserve the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression does not alter the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression maintains the tumor-derived TCR reservoir.

In some implementations, temporary alteration of a protein induces a change in the expression of a specific gene. In some implementations, the temporary alteration of protein expression targets genes including (but not limited to) the following: PD-1 (also known as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, chimeric co-stimulatory receptor (CCR), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, and CC. R5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some implementations, protein expression targeting genes selected from the following groups is temporarily altered: PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL. 2(MCP-1), CCL3(MIP-1α), CCL4(MIP1-β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some embodiments, protein expression is temporarily altered to target PD-1. In some embodiments, protein expression is temporarily altered to target TGFBR2. In some embodiments, protein expression is temporarily altered to target CCR4/5. In some embodiments, protein expression is temporarily altered to target CBL-B. In some embodiments, protein expression is temporarily altered to target CISH. In some embodiments, protein expression is temporarily altered to target CCR (chimeric co-stimulatory receptor). In some embodiments, protein expression is temporarily altered to target IL-2. In some embodiments, protein expression is temporarily altered to target IL-12. In some embodiments, protein expression targeting IL-15 is temporarily altered. In some embodiments, protein expression targeting IL-21 is temporarily altered. In some embodiments, protein expression targeting NOTCH 1/2 ICD is temporarily altered. In some embodiments, protein expression targeting TIM3 is temporarily altered. In some embodiments, protein expression targeting LAG3 is temporarily altered. In some embodiments, protein expression targeting TIGIT is temporarily altered. In some embodiments, protein expression targeting TGFβ is temporarily altered. In some embodiments, protein expression targeting CCR1 is temporarily altered. In some embodiments, protein expression targeting CCR2 is temporarily altered. In some embodiments, protein expression targeting CCR4 is temporarily altered. In some embodiments, protein expression targeting CCR5 is temporarily altered. In some embodiments, protein expression targeting CXCR1 is temporarily altered. In some embodiments, protein expression targeting CXCR2 is temporarily altered. In some embodiments, protein expression targeting CSCR3 is temporarily altered. In some embodiments, protein expression targeting CCL2 (MCP-1) is temporarily altered. In some embodiments, protein expression targeting CCL3 (MIP-1α) is temporarily altered. In some embodiments, protein expression targeting CCL4 (MIP1-β) is temporarily altered. In some embodiments, protein expression targeting CCL5 (RANTES) is temporarily altered. In some embodiments, protein expression targeting CXCL1 is temporarily altered. In some embodiments, protein expression targeting CXCL8 is temporarily altered. In some embodiments, protein expression targeting CCL22 is temporarily altered. In some embodiments, protein expression targeting CCL17 is temporarily altered. In some embodiments, protein expression targeting VHL is temporarily altered. In some embodiments, protein expression targeting CD44 is temporarily altered. In some embodiments, protein expression targeting PIK3CD is temporarily altered. In some embodiments, protein expression targeting SOCS1 is temporarily altered. In some embodiments, protein expression targeting thymocyte selection-associated high migration group (HMG) box (TOX) is temporarily altered. In some embodiments, protein expression targeting ankyrin repeat domain 11 (ANKRD11) is temporarily altered. In some embodiments, protein expression targeting BCL6 co-inhibitor (BCOR) is temporarily altered. In some embodiments, protein expression targeting cAMP protein kinase A (PKA) is temporarily altered.

In some embodiments, a temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors. In some embodiments, the chemokine receptors overexpressed due to temporary protein expression include receptors with ligands, said ligands including (but not limited to) CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.

In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and/or TGFβ (including resulting in, for example, TGFβ pathway blockade). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CBLB (CBL-B). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CISH. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of TIGIT. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors to, for example, improve TIL transport or motility to tumor sites. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CCRs (chimeric co-stimulatory receptors). In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors selected from the group consisting of: CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.

In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins. In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins selected from the group consisting of IL-2, IL-12, IL-15, and/or IL-21.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of NOTCH 1/2 ICD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of VHL. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CD44. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of PIK3CD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of SOCS1.

In some implementations, temporarily altering protein expression causes a decrease and/or reduction in the expression of cAMP protein kinase A (PKA).

In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of two molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one molecule selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of the following: PD-1, CTLA-4, LAG-3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one of the following: CTLA-4, LAG3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CTLA-4. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIM-3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and PD-1. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and TIGIT.

In some implementations, adhesion molecules selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof are inserted into a first TIL population, a second TIL population, or a collected TIL population via gamma retrovirus or lentivirus methods (e.g., increased expression of adhesion molecules).

In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBL-B, and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, expression is reduced by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some implementations, expression is reduced by at least approximately 90%. In some implementations, expression is reduced by at least approximately 95%. In some implementations, expression is reduced by at least approximately 99%.

In some embodiments, expression is increased by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some implementations, expression increases by at least about 90%. In some implementations, expression increases by at least about 95%. In some implementations, expression increases by at least about 99%.

In some embodiments, the temporary alteration of protein expression is induced by treating the TIL with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in the TIL. In some embodiments, intracellular delivery of transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression is performed using a microfluidic platform without SQZ vectors. Such methods have demonstrated the ability to deliver proteins comprising transcription factors to a variety of primary human cell types, including T cells, as described in U.S. Patent Application Publications Nos. US2019/0093073A1, US2018/0201889A1, and US2019/0017072A1, the disclosures of which are each incorporated herein by reference. Such methods can be used in this invention to expose a TIL population to transcription factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression provide an increase in the expression of tumor antigens and/or the number of tumor antigen-specific T cells in the TIL population, thereby leading to TIL population reprogramming and increased therapeutic efficacy of the reprogrammed TIL population compared to the non-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in effector T cells and/or central memory T cell subsets relative to the initial or previous TIL population (i.e., prior to reprogramming), as described herein.

In some embodiments, the transcription factor (TF) includes (but is not limited to) TCF-1, NOTCH 1/2 ICD, and/or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1/2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered with an induced pluripotent stem cell culture (iPSC), such as a commercially available KNOCKOUT serum alternative (Gibco/ThermoFisher), to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered with an iPSC mixture to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is not administered with an iPSC mixture. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some implementations, reprogramming causes a percentage increase in TSCM of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% of TSCM.

In some embodiments, the method of temporarily altering protein expression as described above may be combined with a method of genetically modifying TIL populations, including a step of stabilizing the incorporation of genes for producing one or more proteins. In some embodiments, the method includes a step of genetically modifying TIL populations. In some embodiments, the method includes genetically modifying a first TIL population, a second TIL population, and/or a third TIL population. In some embodiments, the method of genetically modifying TIL populations includes a retroviral transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in: Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71; and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a gamma-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include transposases provided as DNA expression vectors or as expressible RNA or proteins, such that long-term expression of the transposase does not occur in transgenic cells, for example, transposases provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems, including salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, are described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In some implementations, temporary alteration of protein expression in the TIL is induced by small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, which is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where it interferes with the expression of specific genes having complementary nucleotide sequences.

In some implementations, the temporary alteration of protein expression is a reduction in expression. In some implementations, the temporary alteration of protein expression in the TIL is induced by self-delivered RNA interference (sdRNA), which is a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or _-OCH3 ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand bound to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence. sdRNA is a covalently and hydrophobically modified RNAi compound that does not require a delivery medium to enter the cell. sdRNA is typically an asymmetrically modified nucleic acid molecule with a very small double-stranded region. sdRNA molecules typically contain single-stranded and double-stranded regions, and various chemical modifications can be incorporated into these regions. Furthermore, as described herein, sdRNA molecules can be linked to hydrophobic conjugates such as conventional and higher sterol molecules. sdRNAs and related methods for their preparation have been extensively described, for example, in U.S. Patent Application Publications US2016/0304873 A1, US2019/0211337A1, US2009/0131360A1, and US2019/0048341A1, and U.S. Patents 10,633,654 and 10,913,948B2, the disclosures of which are each incorporated herein by reference. To optimize sdRNA structure, chemical properties, target location, sequence preference, etc., an algorithm has been developed and used for sdRNA efficacy prediction. Based on these analyses, a functional sdRNA sequence is generally defined as one whose expression is reduced by more than 70% at a concentration of 1 μM, with a probability exceeding 40%.

Double-stranded DNA (dsRNA) can generally be used to define any molecule containing a pair of complementary RNA strands, typically sense (passenger) and antisense (guide) strands, and may include single-stranded overhang regions. Unlike siRNA, the term dsRNA generally refers to a precursor molecule comprising the sequence of an siRNA molecule, which is released from a larger dsRNA molecule by the action of a lysin system (including Dicer).

In some implementations, the method includes temporarily altering protein expression in a population of TILs (including TILs modified to express CCR), including using self-delivered RNA interference (sdRNA), which is, for example, a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or -OCH ₃ ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand that binds to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Methods using siRNA and sdRNA have been described in the following: Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248; Byrne et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93, the disclosures of which are incorporated herein by reference. In some embodiments, siRNA delivery is accomplished using electroporation or cell membrane disruption (e.g., squeezing or SQZ). In some embodiments, delivery of siRNA or sdRNA to a TIL population does not require electroporation, SQZ, or other methods; the TIL population is actually exposed to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, the method includes delivery of siRNA or sdRNA to a TIL population, which involves exposing the TIL population to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 10 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration between 0.1 μM/10,000 TILs and 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days, wherein the exposure to siRNA or sdRNA is performed two, three, four, or five times by adding fresh siRNA or sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. Patent Application Publications Nos. US 2011/0039914A1, US 2013/0131141A1 and US 2013/0131142A1, and U.S. Patent No. 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA is inserted into the TIL population during manufacturing. In some embodiments, the sdRNA encodes RNA that interferes with the following: NOTCH 1/2 ICD, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and/or CBL-B. In some embodiments, the expression reduction is determined based on the percentage of gene silencing assessed, for example, by flow cytometry and/or qPCR. In some embodiments, the expression reduction is approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

Self-deliverable RNAi technology based on chemically modified siRNA can be used in the methods of this invention to successfully deliver sdRNA to TILs as described herein. The combination of backbone modification with asymmetric siRNA structures and hydrophobic ligands (see, for example, Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93 and U.S. Patent Application Publication No. 2016/0304873A1, the disclosure of which is incorporated herein by reference) allows sdRNA to permeate cultured mammalian cells simply by adding it to the culture medium, utilizing the nuclease stability of sdRNA without the need for additional formulations and methods. This stability allows for the support of constant levels of RNAi-mediated reduction in target gene activity simply by maintaining an effective concentration of sdRNA in the culture medium. Although not bound by theory, backbone stabilization of sdRNA provides extended reductions in gene expression that can persist for months in non-dividing cells.

In some embodiments, over 95% TIL transfection efficiency and target expression reduction occur through various specific siRNAs or sdRNAs. In some embodiments, siRNAs or sdRNAs containing several unmodified ribose residues undergo fully modified sequence substitutions to increase the potency and/or lifetime of the RNAi effect. In some embodiments, the expression reduction effect lasts for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or longer. In some embodiments, the expression reduction effect decreases after siRNA or sdRNA treatment of TILs for 10 days or longer. In some embodiments, target expression is reduced by more than 70%. In some embodiments, target expression in TILs is reduced by more than 70%. In some embodiments, expression reduction in the PD-1/PD-L1 pathway allows TILs to exhibit a more potent in vivo effect, which in some embodiments is due to avoiding the inhibitory effects of the PD-1/PD-L1 pathway. In some embodiments, TIL proliferation is increased due to reduced PD-1 expression by siRNA or sdRNA.

In some embodiments, the sdRNA sequence used in this invention exhibits a 70% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 75% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 80% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 85% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 90% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 95% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 99% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM to about 4 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 4.0 μM.

In some embodiments, the siRNA or sdRNA oligonucleotide agent comprises one or more modifications to increase the stability and/or efficacy of the therapeutic agent and to enable efficient delivery of the oligonucleotide to the cells or tissues to be treated. Such modifications may include 2'-O-methyl modifications, 2'-O-fluorine modifications, dithiophosphate modifications, 2'F-modified nucleotides, 2'-O-methyl-modified and/or 2'-deoxynucleotides. In some embodiments, the oligonucleotide is modified to include one or more hydrophobic modifications, including, for example, sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and/or phenyl. In some embodiments, the chemically modified nucleotide is a combination of thiophosphate, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and thiophosphate. In some embodiments, the sugar may be modified, and the modified sugar may include (but is not limited to) D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halogen (including 2'-fluorine), T-methoxyethoxy, 2'-allyloxy ( _-OCH₂CH = _CH₂ ), 2'-propynyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano. In some embodiments, the sugar moiety may be a hexose and incorporated into an oligonucleotide, such as Augustyns et al., Nucl. Acids. Res. 1992, 18, 4711, the disclosure of which is incorporated herein by reference.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is double-stranded along its entire length, i.e., without a dangling single-stranded sequence at either end of the molecule, i.e., a blunt end. In some embodiments, individual nucleic acid molecules may have different lengths. In other words, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is not double-stranded along its entire length. For example, when using two separate nucleic acid molecules, one of the molecules, such as the first molecule containing an antisense sequence, may be longer than the second molecule with which it hybridizes (leaving a portion of the molecule single-stranded). In some embodiments, when using a single nucleic acid molecule, a portion of the molecule at either end may remain single-stranded.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention contain mismatches and/or loops or protrusions, but are double-stranded for at least about 70% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded for at least about 80% of the oligonucleotide length. In other embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 90%-95% of the oligonucleotide length. In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 96%-98% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention contain at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

In some embodiments, siRNA or sdRNA oligonucleotides may be substantially protected against nucleases, for example, by modifying the 3' or 5' bonds, as described in U.S. Patent No. 5,849,902, the disclosure of which is incorporated herein by reference. For example, oligonucleotides may be made resistant by incorporating a “blocking group.” As used herein, the term “blocking group” refers to a substituent (e.g., other than an OH group) that can be linked to the oligonucleotide or nucleoside as a protecting or coupling group for synthesis (e.g., FITC, propyl ( _CH₂ - _CH₂ - _CH₃ ), diol (-O- _CH₂ - ^CH₂ _- O-) phosphate ( _PO₃²⁻ ), hydrogen phosphonate, or aminophosphite). A “blocking group” may also include a “terminal blocking group” or “exonuclease blocking group” that protects the 5' and 3' ends of the oligonucleotide, comprising modified nucleotide and non-nucleotide exonuclease resistant structures.

In some implementations, at least a portion of the continuous polynucleotides within the siRNA or sdRNA are linked by substituents, such as phosphate thioesters.

In some embodiments, chemical modification may result in at least 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 13% Cellular uptake of siRNA or sdRNA is enhanced at 0%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, multiple C and U residues contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U may contain hydrophobic modifications. In some embodiments, all C and U contain hydrophobic modifications.

In some embodiments, siRNA or sdRNA molecules exhibit enhanced endosome release via the incorporation of a protonable amine. In some embodiments, the protonable amine is incorporated into the sense strand (the molecular portion discarded after RISC loading). In some embodiments, the siRNA or sdRNA compounds of the present invention comprise an asymmetric compound comprising a double-stranded region (required for efficient RISC entry, 10-15 bases long) and a single-stranded region 4-12 nucleotides long; a double-stranded region having 13 nucleotides. In some embodiments, a single-stranded region of 6 nucleotides is employed. In some embodiments, the single-stranded region of the siRNA or sdRNA comprises 2-12 phosphate thioester nucleotide links (referred to as phosphate thioester modification). In some embodiments, 6-8 phosphate thioester nucleotide links are employed. In some embodiments, the siRNA or sdRNA compounds of the present invention further include unique chemical modification patterns that provide stability and compatibility with RISC entry. For example, the guide strand can also be modified by any chemical modification that demonstrates stability without interfering with RISC entry. In some implementations, the chemical modification pattern in the guide chain includes 2'F modification of most C and U nucleotides and phosphorylation at the 5' end.

In some embodiments, at least 30% of the nucleotides in the siRNA or sdRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6 3%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides are modified. In some embodiments, 100% of the nucleotides in the siRNA or sdRNA are modified.

In some embodiments, the siRNA or sdRNA molecule has a very small double-stranded region. In some embodiments, the double-stranded region of the molecule is between 8 and 15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides in length. There may be 100% complementarity between the guide strand and the passenger strand, or one or more mismatches may exist between the guide strand and the passenger strand. In some embodiments, the molecule has a blunt end or a dangling structure with one nucleotide at one end. The single-stranded region of the molecule is between 4 and 12 nucleotides in length in some embodiments. In some embodiments, the single-stranded region may be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In some embodiments, the single-stranded region may also be less than 4 nucleotides or greater than 12 nucleotides in length. In some embodiments, the single-stranded region is 6 or 7 nucleotides in length.

In some embodiments, the siRNA or sdRNA molecules exhibit increased stability. In some cases, the chemically modified siRNA or sdRNA molecules have a half-life in culture medium longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours, including any intermediate values. In some embodiments, the siRNA or sdRNA has a half-life in culture medium exceeding 12 hours.

In some embodiments, siRNA or sdRNA is optimized to increase efficacy and/or reduce toxicity. In some embodiments, the nucleotide length of the guide strand and/or passenger strand and/or the number of phosphate thioester modifications in the guide strand and/or passenger strand can affect the efficacy of the RNA molecule in some respects, while replacing 2'-fluorine (2'F) modifications with 2'-O-methyl (2'OMe) modifications can affect the toxicity of the molecule in some respects. In some embodiments, reducing the 2'F content of the molecule is expected to reduce the toxicity of the molecule. In some embodiments, the number of phosphate thioester modifications in the RNA molecule can affect the efficiency of molecule uptake into cells, for example, passive uptake of molecules into cells. In some embodiments, siRNA or sdRNA does not have 2'F modifications, but is characterized by equivalent efficacy in cellular uptake and tissue penetration.

In some embodiments, the guide chain is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate ester modifications. For example, the guide chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate ester-modified nucleotides. The guide chain may contain one or more modifications that confer increased stability without interfering with RISC entry. Phosphate-ester-modified nucleotides, such as thiophosphate-modified nucleotides, may be at the 3' end, 5' end, or throughout the entire guide chain. In some embodiments, the 3' end 10 nucleotides of the guide chain contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 thiophosphate-modified nucleotides. The guide chain may also contain 2'F and/or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at position 1 in the guide chain (the nucleotide at the 5' end of the guide chain) is modified with 2'OMe and/or phosphorylated. C and U nucleotides within the guide chain may be modified with 2'F. For example, the C and U nucleotides at positions 2-10 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'F. The C and U nucleotides within the guide chain may also be modified with 2'OMe. For example, the C and U nucleotides at positions 11-18 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'OMe. In some embodiments, the nucleotides at the 3' end of the guide chain are unmodified. In some embodiments, most of the C and U nucleotides within the guide chain are modified with 2'F, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, the 5' end of the guide chain is phosphorylated, and the C or U at positions 2-10 are modified with 2'F.

Self-deliverable RNAi technology provides a method for directly transfecting cells with an RNAi agent (whether siRNA, sdRNA, or other RNAi agents) without additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity, and ease of use are characteristics of the compositions and methods, which offer significant functional advantages over conventional siRNA-based techniques, and therefore the sdRNA method is employed in several embodiments of the method for reducing target gene expression in TILs of the present invention. The sdRNA method allows for the direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues in vitro and in vivo. The sdRNAs described in some embodiments of the invention herein are available from Advirna LLC, Worcester, MA, USA.

siRNA and sdRNA can be formed by hydrophobically modified siRNA-antisense oligonucleotide hybridization and are disclosed, for example, in Byrne et al., J. Ocular Pharmacol. Therapeut., 2013, 29, 855-864, the contents of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA oligonucleotides may be delivered to the TILs described herein using sterile electroporation. In some embodiments, the method includes sterile electroporation of TIL populations to deliver siRNA or sdRNA oligonucleotides.

In some embodiments, the oligonucleotide may be delivered to cells in combination with a transmembrane delivery system. In some embodiments, this transmembrane delivery system includes lipids, viral vectors, etc. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that does not require any delivery agent. In some embodiments, the method includes using a transmembrane delivery system to deliver siRNA or sdRNA oligonucleotides to a TIL population.

The oligonucleotides and oligonucleotide compositions are contacted (e.g., brought into contact, also referred to herein as being administered or delivered to) and taken up, including passively via the TIL. sdRNA may be added to the TIL as described herein at the following times: during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D), after step D and before collection in step E, during or after collection in step F, before or during final formulation and/or transfer to the infusion bag in step F, and before any optional cryopreservation step in step F. Furthermore, sdRNA may be added after thawing from any cryopreservation step in step F. In some embodiments, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TIL and other agents at concentrations selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM. In some implementations, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TILs and other agents in amounts selected from the group consisting of: 0.1 μM sdRNA/10,000 TILs/100 μL of medium, 0.5 μM sdRNA/10,000 TILs/100 μL of medium, 0.75 μM sdRNA/10,000 TILs/100 μL of medium. IL/100μL medium, 1μM sdRNA/10,000 TILs/100μL medium, 1.25μM sdRNA/10,000 TILs/100μL medium, 1.5μM sdRNA/10,000 TILs/100μL medium, 2μM sdRNA/10,000 TILs/100μL medium, 5μM sdRNA/10,000 TILs/100μL medium, or 10μM sdRNA/10,000 TILs/100μL medium. In some implementations, one or more sdRNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to TIL cultures twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the pre-REP or REP phase.

The oligonucleotide compositions of the present invention, including siRNA or sdRNA, can be contacted with TILs as described herein during an amplification process, for example, by dissolving high concentrations of siRNA or sdRNA in a cell culture medium and allowing sufficient time for passive uptake. In some embodiments, the method of the present invention includes contacting a TIL population with the oligonucleotide composition as described herein. In some embodiments, the method includes dissolving an oligonucleotide (e.g., siRNA or sdRNA) in a cell culture medium and contacting the cell culture medium with the TIL population. TILs may be a first, second, and/or third population as described herein.

In some embodiments, delivery of oligonucleotides into cells can be enhanced by suitable methods known in the art, including calcium phosphate, DMSO, glycerol or polydextrose, electroporation, or by transfection, for example using cationic, anionic or neutral lipid compositions or liposomes, using methods known in the art, such as those described in: U.S. Patent Nos. 4,897,355; 5,459,127; 5,631,237; 5,955,365; 5,976,567; 10,087,464; and 10,155,945; and Bergan et al., Nucl. Acids Res. 1993, 21,3567, the disclosures of which are each incorporated herein by reference.

In some embodiments, more than one siRNA or sdRNA is used to reduce the expression of a target gene. In some embodiments, one or more of PD-1, TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH that target siRNA or sdRNA are used together. In some embodiments, PD-1 siRNA or sdRNA is used with one or more of TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH to reduce the expression of more than one gene target. In some embodiments, LAG3 siRNA or sdRNA is used in combination with CISH that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, TIGIT siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CISH siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CTLA-4 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, LAG-3 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, CBL-B siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, the siRNA or sdRNA targeting one or more of PD-1, TIM-3, CBL-B, LAG3, and/or CISH described herein is available from Advirna LLC, Worcester, Massachusetts, USA, or from several other suppliers.

In some embodiments, siRNA or sdRNA targets genes selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets genes selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, the siRNA or sdRNA targets genes selected from PD-1, LAG-3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from PD-1 and one of the following: LAG3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets CISH, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets PD-1. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CBL-B.

As discussed herein, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been genetically modified by gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing of TIL populations via nucleotide insertion (RNA or DNA) to promote and inhibit the expression of one or more proteins, and combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into therapeutic populations, wherein the methods include gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, said gene editing techniques being suitable for use according to the present invention. Such methods include those described below as well as viral and transposon methods described elsewhere herein. In some embodiments, methods for genetically modifying TILs, MILs, or PBLs to express CCRs may further include modifications to inhibit gene expression via stable gene knockout or temporary gene knockdown of such genes.

In some embodiments, the method includes methods for genetically modifying TIL populations in a first, second, and/or third population as described herein. In some embodiments, the method for genetically modifying TIL populations includes a step of stabilizing a gene incorporated for producing or repressing (e.g., silencing) one or more proteins. In some embodiments, the method for genetically modifying TIL populations includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, including the step of applying a series of at least three DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time, and such that the survival rate of the TIL is maintained. In some embodiments, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for genetically modifying TIL populations include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population includes a transfection step using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference. The TIL may be a first TIL population, a second TIL population, and/or a third TIL population as described herein.

According to one embodiment, gene editing methods may include the use of programmable nucleases that mediate the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the product of the target gene.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems (e.g., Cas9) target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, the method for amplifying TILs into a therapeutic population can be performed according to any embodiment of the method described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein the method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by in vitro comparison of gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs. In some implementations, the method includes gene editing of TIL populations using CRISPR, TALE, and/or ZFN methods.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs using a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf1). According to a particular embodiment, the CRISPR method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the CRISPR method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

CRISPR stands for clustered regularly spaced short palindromic repeats. The method of gene editing using the CRISPR system is also referred to herein as the CRISPR method. There are three types of incorporating RNA and Cas proteins and CRISPR systems that can be used according to the present invention: type I, II, and III. Type II CRISPR (using Cas9 as an example) is one of the most fully characterized systems.

CRISPR technology is adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins (including Cas9) to prevent attacks from viruses and other foreign organisms by cleaving and destroying the DNA of invading foreign organisms. CRISPR is a DNA-specialized region with two unique characteristics: nucleotide repeat sequences and spacers. The nucleotide repeat sequences are distributed throughout the CRISPR region, with short foreign DNA segments (spacers) interspersed within the repeat sequences. In the type II CRISPR/Cas system, the spacers are integrated into the CRISPR genomic locus and transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNA (tracrRNA) and guide the Cas proteins to perform sequence-specific cleavage and silence pathogenic DNA. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved protospacer adjacent motif (PAM) sequence containing a dinucleotide upstream of the crRNA-binding region. Thus, the CRISPR/Cas system can retarget to cleave almost any DNA sequence by redesigning the crRNA. The crRNA and tracrRNA in the native system can be simplified to a single guide RNA (sgRNA) of approximately 100 nucleotides for genetic engineering. The CRISPR/Cas system can be directly introduced into human cells by co-delivering plasmids expressing the Cas9 endonuclease and the essential crRNA component. Different Cas protein variants can be used to reduce targeting limitations (e.g., heterologous homologs of Cas9, such as Cpf1).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via CRISPR methods include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. , CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using CRISPR methods and applicable to embodiments of the invention are described in U.S. Patents 8,697,359, 8,993,233, 8,795,965, 8,771,945, 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 8,871,445, 8,906,616, and 8,895,308, the disclosures of which are each incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are available from companies such as GenScript.

In some implementations, genetic modification of the TIL population as described herein can be performed using a CRISPR/Cpf1 system as described in U.S. Patent No. US9790490, the disclosure of which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the TALE method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the TALE method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for transcription activator-like effector protein, which includes transcription activator-like effector nucleases (TALENs). The method of gene editing using the TALE system is also referred to herein as the TALE approach. TALEs are naturally occurring proteins from the plant pathogenic bacterium *Xanthomonas* and contain a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize consecutive DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by a 14–20 base-pair spacer region, pull FokI monomers together to dimerize and generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are each incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a zinc finger or zinc finger nuclease method. According to a particular embodiment, the zinc finger method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the zinc finger method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Individual zinc fingers, exhibiting a conserved ββα conformation, contain approximately 30 amino acids. Several amino acids on the α-helix surface typically contact the 3 bp in the main groove of the DNA at varying levels of selectivity. Zinc fingers possess two protein domains. The first domain is a DNA-binding domain, which includes eukaryotic transcription factors and contains the zinc finger. The second domain is a nuclease domain, which includes the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

Individual zinc finger (ZFN) DNA-binding domains typically contain between three and six individual zinc finger repeats and can each recognize between nine and 18 base pairs. If the zinc finger domains are specific for their intended target sites, even a pair of three-finger ZFNs recognizing a total of 18 base pairs can theoretically target a single locus in the mammalian genome. One method for generating novel zinc finger arrays is to combine smaller zinc finger “modules” with known specificity. The most common module assembly process involves combining three separate zinc fingers, each recognizing a 3-base-pair DNA sequence, to produce a three-finger array recognizing a 9-base-pair target site. Alternatively, selection-based methods, such as oligopool engineering (OPEN), can be used to select new zinc finger arrays from a randomly grouped library that considers context-dependent interactions between neighboring fingers. Engineered zinc fingers are available from Sangamo Biosciences (Richmond, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the zinc finger method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, and C. ASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6 R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the zinc finger method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which can be used according to embodiments of the invention are described in U.S. Patents 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, each of which is incorporated herein by reference.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which may be used according to other embodiments of the invention are described in Beane et al., Mol. Therapy, 2015, 23, 1380-1390, the disclosure of which is incorporated herein by reference.

In some embodiments, TILs are optionally genetically engineered to include other functionalities, including (but not limited to) high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). In some embodiments, the method includes genetically engineering a population of TILs to include high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). Suitablely, the TIL population may be a first, second, and/or third population as described herein.

D. Closed systems used in TIL manufacturing

This invention provides a closed system for use during TIL (Total Intake Liquid) culture. Such a closed system allows for the prevention and/or reduction of microbial contamination, allows for the use of fewer culture flasks, and allows for lower costs. In some embodiments, the closed system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

A sterile connection device (STCD) creates a sterile weld between two compatible tubes. This procedure allows for the aseptic connection of multiple containers and tube diameters. In some embodiments, the closure system includes a Luer lock and a heat-sealing system as described in the examples. In some embodiments, the closure system is introduced under aseptic conditions via a syringe to maintain the sterility and closure properties of the system. In some embodiments, a closure system as described in the examples is used. In some embodiments, the TIL is formulated into a final product formulation container according to the method described in the examples herein.

In some embodiments, the closed system uses a single container from the time the tumor fragment is obtained until it is ready for TIL administration to the patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G container, and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. The closed system or closed TIL cell culture system is characterized by tightly sealing the system from the outside to create a closed environment free from bacterial, fungal, and/or other microbial contamination once the tumor sample and/or tumor fragment has been added.

In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

The closed system allows TILs to grow in the absence of microbial contamination and/or with significantly reduced microbial contamination.

Furthermore, the pH, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment each change with cell culture. Therefore, even if the culture medium suitable for cell culture is circulated, the closed environment still needs to be continuously maintained to provide the optimal environment for TIL proliferation. For this purpose, it is desirable to monitor the physical factors of pH, carbon dioxide partial pressure, and oxygen partial pressure within the culture medium of the closed environment using sensors. These signals are used to control a gas exchanger installed at the inlet of the culture environment, and the gas partial pressure of the closed environment is adjusted in real time according to changes in the culture medium to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system incorporating a gas exchanger equipped with monitoring devices for measuring the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment at the inlet, and optimizing the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring devices.

In some implementations, the pressure within the closed environment is controlled continuously or intermittently. That is, the pressure within the closed environment can be varied by means of, for example, a pressure maintaining device, thereby ensuring that the space is suitable for TIL growth under positive pressure or promoting exudation of fluid and thus promoting cell proliferation under negative pressure. Furthermore, by intermittently applying negative pressure, it is possible to uniformly and effectively displace the circulating fluid within the closed environment by temporarily reducing its volume.

In some implementations, the optimal culture components for TIL proliferation may be replaced or added, and factors including, for example, IL-2 and/or OKT3, and combinations thereof may be added.

E.TIL's optional cryopreservation

Optionally, the primary TIL population (e.g., a second TIL population) or an amplified TIL population (e.g., a third TIL population) can be cryopreserved. In some embodiments, therapeutic TIL populations are cryopreserved. In some embodiments, cryopreservation occurs on TILs collected after a second amplification. In some embodiments, TILs are cryopreserved in exemplary step F of Figures 1 and/or 8 (particularly, e.g., Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs are cryopreserved in infusion bags. In some embodiments, TILs are cryopreserved before being placed in infusion bags. In some embodiments, TILs are cryopreserved without being placed in infusion bags. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is typically accomplished by placing a population of TILs in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are then placed in cryopreservation vials and stored at -80°C for 24 hours, with optional transfer to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some embodiments, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLifeSolutions). In some embodiments, the TIL population is cryopreserved using cryopreservation medium containing dimethyl sulfoxide (DMSO). In some embodiments, the TIL population is cryopreserved using a 1:1 (vol:vol) ratio of CS10 to cell culture medium. In some embodiments, the TIL population is cryopreserved using approximately a 1:1 (vol:vol) ratio of CS10 to cell culture medium (also containing additional IL-2).

As discussed above and illustrated in steps A through E in Figures 1 and/or 8 (especially, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P), cryopreservation can occur at multiple points in the TIL amplification process. In some embodiments, the expanded TIL population after the first amplification (as provided, for example, according to step B) or after one or more second amplifications according to step D of Figure 1 or Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be cryopreserved. Cryopreservation is typically accomplished by placing the TIL population in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are placed in cryopreservation vials and stored at -80°C for 24 hours, wherein optionally they are transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, TILs are cryopreserved according to the method provided in Example 6.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some cases, the TIL populations of step B in Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be immediately cryopreserved using the schemes discussed below. Alternatively, the main TIL population may undergo steps C and D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), and then cryopreservation following step D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Similarly, in cases where genetically modified TILs will be used in a therapy, the TIL population from step B or step D of Figure 1 or Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) may undergo genetic modification for appropriate treatment.

F. Phenotypic characteristics of amplified TILs

In some embodiments, the expression of TILs after amplification is analyzed using multiple phenotypic markers, including those described herein and in the examples. In some embodiments, the expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characterization of TILs is analyzed after the first amplification in step B of FIG1 or FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transition in step C from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transformation from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and after cryopreservation. In some implementations, the phenotypic characteristics of TIL are analyzed after the second amplification in step D from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed after two or more amplifications from step D of FIG1 or FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P).

In some implementations, the marker is selected from the group consisting of CD8 and CD28. In some implementations, the expression of CD8 is examined. In some implementations, the expression of CD28 is examined. In some implementations, compared to other processes (e.g., the Gen 3 process provided in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B, the expression of CD28 is examined. The 2A process provided in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) produces a higher expression of CD8 and/or CD28 on the TIL according to the process of the present invention. In some implementations, the expression of CD8 on the TIL produced by the process according to the invention is higher than that of other processes (e.g., the Gen 3 process provided in, for example, FIG8 (especially, for example, FIG8B)) and the 2A process provided in, for example, FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, the expression of CD28 on the TIL produced by the process according to the invention is higher than that of the 2A process provided, such as, for example, in Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to other processes (e.g., Figure 8A and/or Figure 2A, for example, Figure 8A). In some embodiments, high CD28 expression indicates a younger and more durable TIL phenotype. In some embodiments, the expression of one or more regulatory markers is measured.

In some implementations, during any step of the method for amplifying tumor-infiltrating lymphocytes (TILs) as described herein, no selection of a first TIL population, a second TIL population, a third TIL population, or a collected TIL population is made based on CD8 and/or CD28 expression.

In some embodiments, the percentage of central memory cells in the TIL produced by the method according to the invention is higher than that of process 2A, as provided in, for example, Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the memory markers of the central memory cells are selected from the group consisting of CCR7 and CD62L.

In some implementations, the CD4+ and/or CD8+ TIL memory subsets may be divided into different memory subsets. In some implementations, the CD4+ and/or CD8+ TILs contain initial (CD45RA+CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain central memory (CM; CD45RA-CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain effect memory (EM; CD45RA-CD62L-) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain RA+ effect memory/effect (TEMRA/TEFF; CD45RA+CD62L+) TILs.

In some embodiments, the TIL expresses one or more markers selected from the group consisting of granzyme B, perforin, and granulosin. In some embodiments, the TIL expresses granzyme B. In some embodiments, the TIL expresses perforin. In some embodiments, the TIL expresses granulosin.

In some embodiments, a cytokine release assay may also be used to evaluate cytokine release from restimulated TILs. In some embodiments, interferon-γ (IFN-γ) secretion from TILs may be evaluated. In some embodiments, IFN-γ secretion is measured by an ELISA assay. In some embodiments, IFN-γ secretion is measured by an ELISA assay after a rapid second amplification step, following step D as provided, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL health is measured by IFN-γ (IFN-γ) secretion. In some embodiments, IFN-γ secretion indicates active TILs. In some embodiments, a potency assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the culture medium of TILs stimulated with antibodies against CD3, CD28, and CD137/4-1BB. The IFN-γ level in the culture medium from these stimulated TILs can be determined by measuring IFN-γ release. In some embodiments, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), step D in the Gen 3 process provided in the TIL shows increased IFN-γ production compared to step D in the 2A process provided, for example, Figure 8 (especially, for example, Figure 8A), indicating an increased cytotoxic potential of the TIL in step D. In some embodiments, IFN-γ secretion is increased by one, two, three, four, or five times or more. In some embodiments, IFN-γ secretion is increased by one time. In some embodiments, IFN-γ secretion is increased by two times. In some embodiments, IFN-γ secretion is increased by three times. In some embodiments, IFN-γ secretion is increased by four times. In some embodiments, IFN-γ secretion is increased by five times. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ in ex vivo TILs is measured. In some embodiments, IFN-γ in ex vivo TILs is measured, including TILs produced by the methods of the present invention (including, for example, the method of Figure 8B).

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL to about 1000 pg/mL or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some implementations, it is capable of secreting at least 200 pg/mL, at least 250 pg/mL, at least 300 pg/mL, at least 350 pg/mL, at least 400 pg/mL, at least 450 pg/mL, at least 500 pg/mL, at least 550 pg/mL, at least 600 pg/mL, at least 650 pg/mL, at least 700 pg/mL, at least 750 pg/mL, at least 800 pg/mL, at least 850 pg/mL. L, at least 900 pg/mL, at least 950 pg/mL, or at least 1000 pg/mL or more of IFN-γ TILs are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL/5e5 cells to about 1000 pg/mL/5e5 cells or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, it is capable of secreting at least 200 pg/mL/5e5 cells, at least 250 pg/mL/5e5 cells, at least 300 pg/mL/5e5 cells, at least 350 pg/mL/5e5 cells, at least 400 pg/mL/5e5 cells, at least 450 pg/mL/5e5 cells, at least 500 pg/mL/5e5 cells, at least 550 pg/mL/5e5 cells, at least 600 pg/mL/5e5 cells, at least 650 pg/mL/5e5 cells, at least 700 pg/mL/5e5 cells, and at least 750 pg/mL/5e5 cells. TILs of IFN-γ produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) are TILs produced by at least 800 pg/mL/5e5 cells, at least 850 pg/mL/5e5 cells, at least 900 pg/mL/5e5 cells, at least 950 pg/mL/5e5 cells, or at least 1000 pg/mL/5e5 cells or more. In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that express and increase the diversity of T cell reservoirs. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method called Gen2 exemplified in Figure 8 (particularly, for example, Figure 8A). In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased. In some embodiments, processes as described herein (e.g., the Gen 3 process) exhibit higher clonal diversity based on the number of unique peptide CDRs within the sample compared to other processes (e.g., the Gen 2 process).

In some embodiments, TIL activation and depletion can be determined by examining one or more biomarkers. In some embodiments, activation and depletion can be determined using multicolor flow cytometry. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: CD3, PD-1, 2B4/CD244, CD8, CD25, BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183, CD69, CD95, CD127, CD103, and/or LAG-3. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, PD-1, TIGIT, and/or TIM-3. In some embodiments, activation and depletion of markers include (but are not limited to) one or more markers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, CD103+/CD69+, CD103+/CD69-, PD-1, TIGIT, and/or TIM-3. In some embodiments, T cell markers (including activation and depletion markers) may be identified and/or analyzed to examine T cell activation, suppression, or function. In some embodiments, T cell markers may include (but are not limited to) one or more markers selected from the group consisting of: TIGIT, CD3, FoxP3, Tim-3, PD-1, CD103, CTLA-4, LAG-3, BTLA-4, ICOS, Ki67, CD8, CD25, CD45, CD4, and/or CD59.

In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, TILs exhibiting secretion of granzyme B at levels higher than 1000 pg/mL to 300000 pg/mL or more are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed at levels above 1000 pg/mL, above 2000 pg/mL, above 3000 pg/mL, above 4000 pg/mL, above 5000 pg/mL, above 6000 pg/mL, above 7000 pg/mL, above 8000 pg/mL, above 9000 pg/mL, above 10000 pg/mL, above 20000 pg/mL, above 30000 pg/mL, above 40000 pg/mL, above 50000 pg/mL, and above 60000 pg/mL. TILs with granzyme B concentrations of 0000 pg/mL, above 70000 pg/mL, above 80000 pg/mL, above 90000 pg/mL, above 100000 pg/mL, or more are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 1000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 2000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 3000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 4000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 5000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 6000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 7000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 8000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 9000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 10,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 20,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 30,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 40,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 50,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 60,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 70,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 80,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 90,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 100,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of granzyme B above 260,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, the amplification method of the present invention produces an amplified TIL population exhibiting increased in vitro granzyme B secretion compared to a non-amplified TIL population, including, for example, the TILs provided in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased by at least one to fifty times or more compared to a non-amplified TIL population. In some embodiments, IFN-γ secretion is increased by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least thirty, at least forty, at least fifty times or more compared to a non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least doubled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least quadrupled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least five times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least six times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least seven times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least eight times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least nine-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least ten-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least twenty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least thirty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least forty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least fifty-fold compared to the non-amplified TIL population.

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times lower levels of TNF-α (i.e., TNF-alpha) compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α (i.e., TNF-alpha) are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, IFN-γ and granzyme B levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, granzyme B and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ, granzyme B, and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some implementations, phenotypic characteristics are examined after cryopreservation.

G. Other process implementation plans

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days or about 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days or about 1 to 10 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling-up of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein in the second container, the second TIL population from the small-scale culture is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days or approximately 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring and distributing the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 to 7 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for approximately 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for approximately 1 to 8 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a vertical scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 5 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a longitudinal scaling up of the culture by: (1) performing rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-g500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by contacting a first TIL population with a culture medium that also contains exogenous antigen-presenting cells (APCs), wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the culture medium is supplemented with additional exogenous APC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 2:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, or 2.3:1. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the initial first amplification is exactly or approximately 1× ^10⁸ , ^1.1 ×10⁸, 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, 1.5× ^10⁸ , 1.6 ^{×10⁸ ,} 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9×10⁸, 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , 3.1× ^10⁸ , or 3.1×10⁸. ⁸ , 3.2× ^10⁸ , 3.3× ^10⁸ , 3.4× ^10⁸ , or 3.5× ^10⁸ APCs, wherein the number of APCs added in the rapid second amplification is exactly or approximately 3.5× ^10⁸ , 3.6×10⁸, ^3.7 × ^10⁸ , 3.8× ^10⁸ , 3.9× ^10⁸ , 4×10⁸, 4.1× ^10⁸ , ^4.2 × ^10⁸ , 4.3× ^10⁸ , 4.4× ^10⁸ , 4.5×10⁸, 4.6× ^10⁸ , 4.7× ^10⁸ , 4.8× ^10⁸ , 4.9× ^10⁸ , 5× ^10⁸ , 5.1× ^10⁸ , 5.2× ^{10⁸ ,} or 5.3× ^10⁸. 5.4× ^10⁸ , 5.5× ^10⁸ , 5.6×10⁸, 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2×10⁸, 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , ^6.6 × ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , ^7.1 × ^10⁸ , 7.2×10⁸, 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , ^7.8 × ^10⁸ 7.9× ^10⁸ , 8× ^10⁸ , 8.1×10⁸, 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7×10⁸, 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , ^9.6 × ^10⁸ , 9.7× ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or} 1× ^10⁹ APCs.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1 × ^10⁸ APCs to exactly or about 3.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 3.5 × ^10⁸ APCs to exactly or about 1 × ^10⁹ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 2 × ^10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or approximately 2.5 × ^10⁸ APCs are added to an initial first amplification, and exactly or approximately 5 × ^10⁸ APCs are added to a rapid second amplification.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein multiple tumor fragments are distributed into multiple separate containers, in each separate container a first TIL population is obtained in step (a), a second TIL population is obtained in step (b), and a third TIL population is obtained in step (c), and therapeutic TIL populations from the multiple containers in step (c) are combined to produce a collected TIL population from step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of tumors are uniformly distributed into a plurality of separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise at least two separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to twenty separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to fifteen separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to ten separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to five separate containers.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the plurality of separate containers comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first amplification of a first TIL population in step (b) is performed in each container, and a rapid second amplification of a second TIL population generated from such a first TIL population is performed in the same container as in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each of the separate containers includes a first breathable surface area.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein multiple tumor fragments are distributed in a single container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a single container includes a first breathable surface region.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in a second container containing a second breathable surface region.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second container is larger than the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the second breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:9.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:2.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected as exactly or greater. Approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4... 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 1.5:1 to exactly or about 100:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 50:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 25:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 50 times larger in number than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times larger than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is supplemented with additional IL-2 exactly or about 2 days or exactly or about 3 days after the start of the second time period in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, further comprising the step of cryopreserving the collected TIL population in step (d) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, including an additional step (e) after step (d) of transferring the collected TIL population from step (d) to an infusion bag optionally containing HypoThermosol.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, comprising the step of cryopreserving an infusion bag containing the collected TIL population in step (e) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation process is performed using a 1:1 ratio of collected TIL populations to cryopreservation medium.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (b) is 2.5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (c) is 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a PBMC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are artificial antigen-presenting cells.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a membrane-based cell processing system is used for the collection in step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the collection in step (d) is performed using the LOVO cell processing system.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 5 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 10 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 15 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 20 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 25 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 30 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 35 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 40 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 45 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or about 50 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 segments per container.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately 27 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 20 ^mm³ to exactly or about 50 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 21 ^mm³ to exactly or about 30 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 22 ^mm³ to exactly or about 29.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 23 ^mm³ to exactly or about 29 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 24 ^mm³ to exactly or about 28.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 25 ^mm³ to exactly or about 28 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 26.5 ^mm³ to exactly or about 27.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately the following: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 30 to exactly or about 60 segments, wherein the total volume is exactly or about 1300 ^mm³ to exactly or about 1500 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total volume is exactly or about 1350 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total mass is exactly or about 1 gram to exactly or about 1.5 grams.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is provided in a container in the form of a G container or a Xuri cell bag.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is from about 10,000 IU/mL to about 5,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is about 6,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second time period in step (c) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 5 days, 6 days, or 7 days, respectively.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 16 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 17 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 16 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 14 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 15 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 14 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 15 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 14 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 15 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 16 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 18 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the therapeutic TIL population collected in step (d) contains a therapeutically effective dose of TIL sufficient for use with TIL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of TILs sufficient for a therapeutically effective dose is exactly or about 2.3 × 10¹⁰ to exactly or about 13.7 × 10¹⁰.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein effector T cells and/or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression relative to effector T cells and/or central memory T cells obtained from the second cell population in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a closed container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a G container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-100.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-500.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared by the methods described in any of the foregoing paragraphs as applicable above.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs) or OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of antigen-presenting cells (APCs) and OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 16 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 17 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 18 days.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased interferon-γ production.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased polyclonalness.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased efficacy.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A through F above, or according to steps A through F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than one times the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than twice the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least three times more interferon-γ.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any antigen-presenting cells (APCs). In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are made capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that is capable of producing at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are able to produce at least one times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least three times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy), a core needle biopsy, a core needle puncture biopsy, or a fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a fine-needle aspirate.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is obtained by core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirates; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first amplification step; (iii) the method includes a period of approximately 8 days for initiating the first amplification; and (iv) the method includes a period of approximately 11 days for rapid second amplification. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating the first expansion step; (iii) the method includes a period of approximately 8 days for initiating the first expansion; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing the subcultures for approximately 6 days. In some of the foregoing embodiments, up to 5 subcultures are cultured separately in containers of the same size or larger as the container in which the second TIL population is initially cultured in the rapid second expansion. In some of the foregoing embodiments, the culture of the second TIL population is evenly distributed among up to 5 subcultures. In some of the aforementioned embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1 to about 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step to obtain a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days; and (iv) the method includes a rapid second expansion step by culturing the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 11 days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days to obtain a second TIL population; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing each of the subcultures in a cell culture medium containing IL-2 for approximately 6 days. In some of the foregoing embodiments, up to five subcultures are cultured in containers of the same size or larger than the container in which the second TIL population was started during the rapid second amplification. In some of the foregoing embodiments, the cultures of the second TIL population are evenly distributed among up to five subcultures. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method comprises obtaining a first TIL population from 1 to about 10 tumor tissue samples from a subject by core needle biopsy; (ii) the method comprises, prior to performing the first amplification step, culturing the first TIL population in a G-REX-100M culture flask in a cell culture medium containing 0.5 L CM1 medium of 6000 IU IL-2/mL for about 3 days; (iii) the method comprises initiating the first amplification by adding 0.5 L CM1 medium containing 6000 IU/mL IL-2, 30 ng/mL OKT-3, and about ^10⁸ feeder cells, and culturing for about 8 days; and (iv) the method comprises performing a rapid second amplification by transferring the second TIL population to a 5 L CM1 medium containing 3000 IU/mL IL-2, 30 ng/mL OKT-3, and 5 × ^10⁹ feeder cells. (a) The culture is divided into up to five subcultures by transferring ^10⁹ TILs into each of up to five G-REX-500MCS culture flasks containing 5 L AIM-V medium with 3000 IU/mL IL-2, and said subcultures are cultured for approximately six days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (a) initiating a first expansion of a first T cell population obtained from a donor by culturing a first T cell population to achieve growth and initiate activation of the first T cell population; (b) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (c) collecting the second T cell population. In other embodiments, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling up of culture scale by: (a) rapidly expanding the first T cell population by culturing it in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second expansion step is broken down into multiple steps to achieve a longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of up to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), the first T cell population is cultured in a first culture medium containing OKT-3 and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises a 4-1BB agonist, OKT-3, and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the first T cell population is cultured in a second culture medium containing OKT-3, IL-2 and antigen-presenting cells (APC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains 4-1BB agonist, OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs in the second APC group to the number of APCs in the first APC group is about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs in the first APC group is about 2.5 × ^10⁸ and the number of APCs in the second APC group is about 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is laminated onto a first breathable surface with an average thickness of two APC layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC group is laminated onto the first breathable surface with an average thickness selected from 4 to 8 APC layers.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the average number of APC layers stacked on the first breathable surface in step (b) to the average number of APC layers stacked on the first breathable surface in step (a) is 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 2.0 × ^10⁶ APCs/ ^cm² to just or about 3.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 2.5 × ^10⁶ APCs/ ^cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 2.0 × ^10⁶ APCs/ ^cm² to exactly or about 3.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a peripheral blood mononuclear cell (PBMC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMCs are irradiated and the donor for the first T cell population is exogenous.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are bone marrow infiltrating lymphocytes (MIL).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are peripheral blood lymphocytes (PBLs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by separation from whole blood of a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by isolating from the donor's apheresis product.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first T cell population is isolated from whole blood or apheresis products of a donor by positive or negative selection of T cell phenotype.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cell phenotype is CD3+ and CD45+.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein T cells are isolated from NK cells prior to initiating a first expansion of a first T cell population. In other embodiments, T cells in a first T cell population are separated from NK cells by removing CD3-CD56+ cells from the first T cell population. In other embodiments, CD3-CD56+ cells are removed from the first T cell population by using a gating strategy that removes CD3-CD56+ cell fractions and recovers negative fractions. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of NK cells. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of CD3-CD56+ cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of NK cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of NK cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with ovarian cancer.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or about 1 × ^10⁷ T cells from a first T cell population are seeded into a container to initiate an initial first expansion culture in such a container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein a first T cell population is distributed into a plurality of containers, and exactly or about 1 × 10⁷ T cells from the first T cell population are seeded in each container to initiate an initial first expansion culture in such containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second T cell population collected in step (c) is a therapeutic TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via small biopsy (including, for example, perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a donor, including small biopsies (such as perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsy of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a donor.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsies, core needle biopsies, or needle biopsies obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the initiation of the first expansion is performed in a container containing a first breathable surface region, wherein the initiation of the first expansion is performed for a first time period of approximately 7 or 8 days to obtain the second TIL population. (iii) A second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iv) a rapid second expansion is performed by supplementing the second TIL population with a second cell culture medium containing additional IL-2, OKT-3 and APC to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (ii), wherein the rapid second expansion is performed for a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container containing a second ventilated surface area; (v) the therapeutic TIL population obtained from step (iii) is collected; and (v) the collected TIL population from step (iv) is transferred to an infusion bag.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsy, core needle biopsy, or needle biopsy samples obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; and (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a second TIL population, wherein the initiation... The first amplification is carried out over a first period of approximately 7 or 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iii) a rapid second amplification is carried out by contacting the second TIL population with a third cell culture medium containing IL-2, OKT-3 and APC to produce a third TIL population, wherein the rapid second amplification is carried out over a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second amplification is carried out in a container containing a second ventilated surface area; and (iv) the therapeutic TIL population obtained from step (iii) is collected.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with an additional amount of third culture medium and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with a fourth medium containing IL-2 and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the culture is split into up to five subcultures after day 5 of the second time period.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein all steps in the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (i) initiating a first expansion of a first T cell population by culturing a first T cell population to achieve growth and initiate activation of the first T cell population, the first T cell population being derived from a tumor sample obtained from one or more small biopsies, core needle biopsies, or needle biopsies of a tumor in a donor; (ii) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population a second T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (iv) collecting the second T cell population. In some embodiments, the tumor sample is obtained from multiple core needle biopsies. In some embodiments, the multiple core needle biopsies are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, and 10 core needle biopsies.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein T cells or TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubating the tumor in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, California). In some embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes one or more dissociation (digestive) enzymes, such as (but not limited to) collagenase (including any blended or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersant enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, protease type XIV (streptomycin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof. In other embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes collagenase (including any blended or type of collagenase), neutral protease (dispersant enzyme), and deoxyribonuclease I (DNase).

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a TIL population undergoes a gene-editing process. In some embodiments, the gene-editing process may be performed at any time during the TIL amplification process, including before, during, or after any step of the amplification process, such as before, during, or after one or more of steps A, B, C, D, E, and F outlined above.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein an activation step follows a first amplification step. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and/or anti-CD38 beads for about 1-7 days. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and anti-CD38 beads for about 1-7 days. In some embodiments, the activation step is performed for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the activation step is followed by a gene editing step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a gene editing step follows a first amplification step. In some embodiments, when the first amplification step includes OKT-3, a gene editing step follows the first amplification step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the gene editing step includes a sterile electroporation step on a TIL population. In some embodiments, the sterile electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates the expression of PD-1. According to some embodiments, the gene editor further comprises a TALE nuclease system that downregulates the expression of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CTLA-4 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/LAG-3 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CISH double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CTLA-4. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and TIGIT. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and TIGIT. According to some implementation schemes, the obtained TILs showed downregulation of CTLA-4 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CISH expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of TIGIT and CBL-B expression.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a quiescent step follows the gene editing step. In some embodiments, the quiescent step comprises incubating a TIL population at about 30-40°C and about 5% _CO2 for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 30°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1 hour, followed by about 15-23 hours at about 30°C.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a second amplification step follows the resting step.

VIII. Pharmaceutical Compositions, Dosage, and Dosing Regimens

In some embodiments, TILs, MILs, or PBLs (including TILs, MILs, or PBLs genetically modified to express CCR) amplified and/or genetically modified using the methods of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC or melanoma. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is metastatic NSCLC or metastatic melanoma. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of NSCLC. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

In other embodiments, the present invention provides an infusion bag that includes the therapeutic TIL population described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a pharmaceutically acceptable carrier as described in any of the preceding paragraphs.

In other embodiments, the present invention provides an infusion bag comprising the TIL composition described in any of the preceding paragraphs.

In other embodiments, the present invention provides a cryopreservation formulation for a therapeutic TIL population as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a cryopreservation culture medium as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains DMSO.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a cryopreservation formulation of the TIL composition described in any of the preceding paragraphs.

In some embodiments, TILs amplified using the methods of this disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly when the cancer is NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

IX. Methods of treating patients

The treatment method begins with the collection and culture of raw TILs. Such methods have been described in the art, for example, by reference in their entirety to Jin et al., J. Immunotherapy, 2012, 35(3):283-292. The implementation of the treatment method is described throughout the following sections, including examples.

The methods described herein, including, for example, those described in steps A through F above or those described in steps A through F above (and also, for example, those shown in Figures 1, 36, and/or 8), have been found to have specific uses in the treatment of cancer patients (e.g., as described in Goff et al., J. Clinical Oncology, 2016, 34(20):2389-239 and the Supplement, which are incorporated herein by reference in their entirety). In some embodiments, TILs are grown from resected metastatic melanoma remnants as previously described (see Dudley et al., J Immunother., 2003, 26:332-342, which are incorporated herein by reference in their entirety). Fresh tumors can be segmented under sterile conditions. Representative samples can be collected for formal pathological analysis. Single fragments of 2 ^mm³ to ³ mm³ can be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained from each patient. In some embodiments, 24 samples are obtained from each patient. Samples can be placed in individual wells of a 24-well plate, maintained in growth medium containing a high dose of IL-2 (6,000 IU/mL), and tumor destruction and/or TIL proliferation can be monitored. As described herein, any tumor cells remaining viable after treatment can be enzymatically digested into a single-cell suspension and cryopreserved.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumors when available. TILs are considered reactive if overnight co-culture produces interferon-γ (IFN-γ) levels >200 pg/mL and twice the background level. (Goff et al., J Immunother., 2010, 33:840-847; incorporated herein by reference in its entirety). In some embodiments, cultures demonstrating autoreactivity or adequate growth patterns may be selected for secondary amplification (e.g., the secondary amplification provided according to step D of Figures 1, 36, and/or 8), including what is sometimes referred to as rapid amplification (REP). In some embodiments, amplified TILs exhibiting high autoreactivity (e.g., high proliferation during secondary amplification) are selected for additional secondary amplification. In some implementations, TILs with high self-reactivity (e.g., high proliferation during the second amplification provided in step D of Figures 1, 36 and/or 8) are selected for additional second amplification according to step D of Figures 1, 36 and/or 8.

Cell phenotypes of cryopreserved samples of infusion bag TILs can be analyzed by flow cytometry targeting surface markers CD3, CD4, CD8, CCR7, and CD45RA (e.g., FlowJo) (BD BioSciences) and by any of the methods described herein. Serum cytokines were measured using standard enzyme-linked immunosorbent assay (ELISA). An increase in serum IFN-γ was defined as a baseline level >100 pg/mL and greater than 43.

In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, achieve remarkable improvements in clinical efficacy. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, exhibit improved clinical efficacy compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8). In some embodiments, methods other than those described herein include methods referred to as Process 1C and/or Gen 1. In some embodiments, the increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1 or 36, exhibit similar response time and safety profiles compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8).

In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of a subject treated with TIL indicates active TIL. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TIL of a subject treated with TIL prepared by the methods of the present invention, including, for example, those described in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TIL produced by the methods of the present invention. In some embodiments, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or compared to patients treated with TIL prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is quadrupled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is increased fivefold compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the serum of subjects treated with TILs prepared using the methods of the present invention, including those prepared, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy in treating tumors.

In some embodiments, the TILs prepared by the methods of the present invention include those described, for example, in Figure 1 or Figure 36, where IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of subjects treated with TILs indicates active TILs. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TILs of subjects treated with TILs prepared by the methods of the present invention, including methods described, for example, in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TILs produced by the methods of the present invention. In some implementations, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, and/or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, and/or 8, such as those referred to as the process 1C method). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, those other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

Measures of efficacy may include disease control rate (DCR) and overall response rate (ORR), as known in the art and described herein.

A. Methods of treating cancer

The compositions and methods described herein can be used in a method of treating a disease. In some embodiments, they are used to treat hyperproliferative conditions, such as cancer, in adult or pediatric patients. They can also be used to treat other conditions as described herein and in the following paragraphs.

In some implementations, hyperplasia is defined as cancer. In some implementations, hyperplasia is defined as solid tumor cancer. In some implementations, solid tumor cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papillomavirus (HPV), central nervous system-related cancers (including ependymoma, neuroblastoma, neuroblastoma, pineal blastoma, and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSC)). C) Hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, and pharyngeal cancer; kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC), metastatic NSCLC, and small cell lung cancer); melanoma (including uveal melanoma, choroidal melanoma, ciliary body melanoma, iris melanoma, or metastatic melanoma); mesothelioma (including malignant pleural mesothelioma); ovarian cancer; pancreatic cancer (including pancreatic duct adenocarcinoma); penile cancer; rectal cancer; kidney cancer; renal cell carcinoma; sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas); thyroid cancer (including anaplastic thyroid cancer); uterine cancer and vaginal cancer.

In some embodiments, the hyperplastic condition is a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from the group consisting of: chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-B, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified MIL or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-BR, wherein the cancer is a hematologic malignancy.

In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, that is relapsed or refractory to treatment with at least one prior therapy (including chemotherapy, radiotherapy, or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least two prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least three prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy).

In some embodiments, the cancer is a microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. MSI-H and dMMR cancers and their detection have been described in Kawakami et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosure of which is incorporated herein by reference.

In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a non-human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a companion animal.

In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors and/or MEK inhibitors. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method of treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, enrafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof; and difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, bemetinib, selmetinib, pimatetinib, refatinib, and pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is a pediatric cancer.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is uveal melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the uveal melanoma is a choroidal melanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is neuroblastoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a central nervous system (CNS) related cancer. In some embodiments, the pediatric cancer is difficult to treat with chemotherapy. In some embodiments, the pediatric cancer is difficult to treat with radiation therapy. In some embodiments, the pediatric cancer is difficult to treat with dinutuximab.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the CNS-related cancer is neuroblastoma, pinealoblastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein can be used to treat cancer that is refractory to anti-PD-1 or anti-PD-L1 antibodies or resistant to prior treatment. In some embodiments, the patient is a primary refractory patient to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has not shown a prior response to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has shown a prior response to anti-PD-1 or anti-PD-L1 antibodies, followed by cancer progression. In some embodiments, the cancer is refractory to combination therapy with anti-CTLA-4 antibodies and/or anti-PD-1 or anti-PD-L1 antibodies in combination with at least one chemotherapeutic agent. In some embodiments, the prior chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and/or cisplatin. In some prior embodiments, the chemotherapeutic agent is a platinum-based dual chemotherapeutic agent. In some embodiments, the platinum dual therapy comprises a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel, docetaxel, or nab-paclitaxel). In some embodiments, the platinum dual chemotherapeutic agent is combined with pemetrexed.

In some implementations, NSCLC is PD-L1 negative and/or comes from patients with cancers that express PD-L1 and have a tumor proportion score (TPS) <1%, as described elsewhere in this article.

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or anti-PD-L1 antibody, pemetrexed, and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some embodiments, NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient has not received treatment. In some embodiments, NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, NSCLC has been previously treated with a chemotherapy agent. In some embodiments, NSCLC has been previously treated with a chemotherapy agent but is no longer being treated with said chemotherapy agent. In some embodiments, the NSCLC patient has not received anti-PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the NSCLC patient has not received NSCLC treatment or has received chemotherapy agent treatment but has not received PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has not received treatment or has received chemotherapy agent treatment but has not received anti-PD-1/PD-L1 treatment and has low PD-L1 expression. In some embodiments, the NSCLC patient had massive disease at baseline. In some embodiments, the subject has massive disease and low PD-L1 expression at baseline. In some embodiments, the NSCLC patient does not have detectable PD-L1 expression. In some embodiments, the NSCLC patient is untreated or has received chemotherapy but not anti-PD-1/PD-L1 treatment and does not have detectable PD-L1 expression. In some embodiments, the patient has massive disease and does not have detectable PD-L1 expression at baseline. In some embodiments, the NSCLC patient has untreated NSCLC or has received chemotherapy (e.g., received chemotherapy agents) but not anti-PD-1/PD-L1 treatment, and the patient has low PD-L1 expression and/or has massive disease at baseline. In some embodiments, massive disease is indicated when the maximum tumor diameter measured in the transverse or coronal plane is greater than 7 cm. In some embodiments, massive disease is indicated when there are swollen lymph nodes with a short axis diameter of 20 mm or greater. In some embodiments, the chemotherapy agents include standard of care treatments for NSCLC.

In some embodiments, PD-L1 expression is determined by a tumor proportion score. In some embodiments, a subject with refractory NSCLC has a tumor proportion score (TPS) of <1%. In some embodiments, a subject with refractory NSCLC has a TPS of ≥1%. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment.

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, or 8, including, for example, methods referred to as Process 1C). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy in cancer treatment. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8).

In some embodiments, PD-L1 expression is determined by tumor proportion scoring using one or more assays as described herein. In some embodiments, the subject or patient with NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the NSCLC tumors have a TPS of ≥1%. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a TPS of ≥1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and a tumor proportion score has been determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment.

In some implementations, NSCLC is NSCLC exhibiting a tumor proportion score (TPS), or the percentage of live tumor cells obtained from the patient prior to anti-PD-1 or anti-PD-L1 therapy, or the percentage of PD-L1 protein showing partial or complete membrane staining at any intensity of less than 1% (TPS<1%). In some implementations, NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, <0.04%, <0.03%, <0.02%, and <0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, and about 0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS between 0% and 1%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.9%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.8%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.7%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.6%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.5%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.4%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.3%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.2%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.1%. TPS can be measured by methods known in the art, such as those described in Hirsch et al., J. Thorac. Oncol. 2017, 12, 208-222, or methods used to determine TPS prior to treatment with pembrolizumab or other anti-PD-1 or anti-PD-L1 therapies. Methods approved by the U.S. Food and Drug Administration for measuring TPS can also be used. In some embodiments, PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

In some embodiments, partial membrane staining includes 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more. In some embodiments, complete membrane staining includes approximately 100% membrane staining.

In some implementations, testing PD-L1 may involve measuring the level of PD-L1 in a patient's serum. In these implementations, measuring PD-L1 in a patient's serum eliminates the uncertainty of tumor heterogeneity and the discomfort of patients undergoing serial biopsies.

In some implementations, elevated soluble PD-L1 levels compared to baseline or standard levels are associated with a prognostic risk of NSCLC progression. See, for example, Okuma et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli et al., Oncotarget, 2018, 9, 17554-17563. In some implementations, PD-L1 is exosomal PD-L1. In some implementations, PD-L1 is expressed on circulating tumor cells.

In some implementation schemes, the subject or patient has non-small cell lung cancer (NSCLC), characterized by at least one of the following:

i. PD-L1 pre-defined tumor proportion score (TPS) <1%,

ii. The TPS score of PD-L1 is 1%-49%, or

iii. The predetermined absence of one or more driver mutations,

The driving mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20 mutations, KRAS mutations, BRAF mutations, ALK mutations, c-ROS mutations (ROS1 mutations), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and/or altered MET signals, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations, and GNA11 mutations.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population described herein.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition described herein.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to the administration of therapeutically effective doses of the therapeutic TIL population and TIL composition described herein, respectively.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, the method further comprising the step of initiating treatment of the subject with a high-dose IL-2 regimen the day after administration of TIL cells to the subject.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg every eight hours via a 15-minute bolus intravenous infusion until tolerated.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is glioblastoma (including GBM).

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides a therapeutic TIL population as described herein in a method for treating a subject suffering from cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer using a TIL composition described herein, comprising administering a therapeutically effective dose of the TIL composition to the subject.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administration of a therapeutically effective dose of the therapeutic TIL population or TIL composition described herein.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, which further includes the step of starting treatment of the patient with a high-dose IL-2 regimen the day after administration of TIL cells.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg via intravenous infusion every eight hours in the form of 15-minute bolus injections until tolerated.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is glioblastoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides the use of the therapeutic TIL population described herein in a method of treating a subject’s cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides the use of the TIL composition described in any of the foregoing paragraphs in a method of treating a subject’s cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In other embodiments, the present invention provides the use of the therapeutic TIL population or the TIL composition described herein in a method of treating a patient with cancer, the method comprising administering a non-myeloablative lymphocyte depletion regimen to the patient and then administering to the subject a therapeutically effective dose of the therapeutic TIL population or the therapeutically effective dose of the TIL composition described herein in any of the preceding paragraphs.

1. Combination with PD-1 and PD-L1 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, which includes TIL and one or more PD-1 and/or PD-L1 inhibitors.

Programmed death 1 (PD-1) is a 288-amino acid transmembrane immune checkpoint receptor protein expressed by T cells, B cells, natural killer (NK) T cells, activated monocytes, and dendritic cells. PD-1, also known as CD279, belongs to the CD28 family and is encoded by the Pdcd1 gene on chromosome 2 in humans. PD-1 consists of an immunoglobulin (Ig) superfamily domain, a transmembrane domain, and an intracellular domain containing the immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoreceptor tyrosine switching motif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play important roles in immune tolerance, as described by Keir et al., Annu. Rev. Immunol. 2008, 26, 677-704. PD-1 provides a suppressive signal that negatively regulates T cell immune responses. PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273) are expressed on tumor cells and stromal cells, which may encounter activated T cells expressing PD-1, leading to immunosuppression of T cells. PD-L1 is a 290-amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9. Blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2 using PD-1 inhibitors, PD-L1 inhibitors, and/or PD-L2 inhibitors can overcome immune resistance, as demonstrated in recent clinical studies, such as those described in Topalian et al., N. Eng. J. Med. 2012, 366, 2443-54. PD-L1 is expressed on many tumor cell lines, while PD-L2 is primarily expressed on dendritic cells and some tumor lines. In addition to T cells (which induce PD-1 expression upon activation), PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes, and dendritic cells.

In some embodiments, the PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail below. Regarding PD-1 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, references herein to PD-1 inhibitors as antibodies may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, references herein to PD-1 inhibitors may also refer to small molecule compounds or their pharmaceutically acceptable salts, esters, solvates, hydrates, cocrystals, or prodrugs.

In some embodiments, the PD-1 inhibitor is an antibody (i.e., an anti-PD-1 antibody) or a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In some embodiments, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competitively binds to PD-1 and/or binds to an epitope on PD-1. In some embodiments, the antibody competitively binds to PD-1 and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a KD of about 100 pM or less, at a KD of about 90 pM or less, at a KD of about 80 pM or less, at a KD of about 70 pM or less, at a KD of about 60 pM or less, at a KD of about 50 pM or less, at a KD of about 40 pM or less, at a KD of about 30 pM or less, at a KD of about 20 pM or less, at a KD of about 10 pM or less, or at a KD of about 1 pM or less.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a k _{-association rate} of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about ⁸ × 10⁵ l/M·s or faster, at a k _{-association rate} of about 8.5 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9.5 × ^10⁵ l/M·s or faster, or at a k- _{association rate} of about 1 × ^10⁶ l/M·s or faster.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, or with a k _{-dissociation rate} of about 2.1 × ^10⁻⁵ 1/s or slower, or with a k-dissociation rate of about 2.2 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.3 × 10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.6 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.7 × ^10⁻⁵ 1/s or slower. It binds to human PD-1 at a k- _{dissociation rate} of 1/s or slower, at a k- _{dissociation rate} of approximately 2.8 × ^10⁻⁵ 1/s or slower, at a k- _{dissociation rate} of approximately 2.9 × ^10⁻⁵ 1/s or slower, or at a k- _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, or about 6 nM or less. The binding of human PD-L2 to human PD-1; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower; or blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-1 inhibitor is nivolumab (available from Bristol-Myers Squibb via OPDIVO) or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-1 receptor. In some embodiments, the anti-PD-1 antibody is an immunoglobulin G4κ anti-(human CD274) antibody. Nivolumab is designated Chemical Abstracts Service (CAS) Registry No. 946414-94-4 and is also known as 5C4, BMS-936558, MDX-1106, and ONO-4538. The preparation and characteristics of nivolumab are described in U.S. Patent No. 8,008,449 and International Patent Publication No. WO 2006/121168, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of nivolumab in various forms of cancer have been described in Wang et al., Cancer Immunol. Res. 2014, 2, 846-56; Page et al., Ann. Rev. Med., 2014, 65, 185-202; and Weber et al., J. Clin. Oncology, 2013, 31, 4311-4318, the contents of which are incorporated herein by reference. The amino acid sequence of nivolumab is presented in Table 18. Nivolumab has intra-heavy chain disulfide bonds at 22-96, 140-196, 254-314, 360-418, 22”-96”, 140”-196”, 254”-314”, and 360”-418”; intra-heavy chain disulfide bonds at 23”-88”, 134”-194”’, 23”’-88”’, and 134”’-194”’; inter-heavy chain disulfide bonds at 127-214”’ and 127”-214”’; inter-heavy chain disulfide bonds at 219-219” and 222-222”; and N-glycosylation sites (H CH2 84.4) at 290” and 290”.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:158 and the light chain provided in SEQ ID NO:159. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO:159, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of nivolumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:160, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:161 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:165, SEQ ID NO:166, and SEQ ID NO:167, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of nivolumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab.

Table 18. Amino acid sequences of PD-1 inhibitors associated with nivolumab.

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at the following doses: about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 240 mg every 2 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 480 mg every 4 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma, with approximately 1 mg/kg of nivolumab administered every 3 weeks on the same day, followed by 3 mg/kg of ipilimumab for 4 consecutive doses, then 240 mg every 2 weeks or 480 mg of nivolumab every 4 weeks.

In some implementations, nivolumab is administered as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks and ipilimumab at approximately 1 mg/kg every 6 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks, plus ipilimumab at 1 mg/kg every 6 weeks and two cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks or 480 mg every 4 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks and ipilimumab at 1 mg/kg every 6 weeks to treat malignant pleural mesothelioma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks and 480 mg of nivolumab every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks to treat pediatric patients <40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 240 mg nivolumab every 2 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 480 mg nivolumab every 4 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab is administered every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 480 mg of nivolumab is administered every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor comprises pembrolizumab (available from Merck Inc. of Kenilworth, NJ, USA under the KEYTRUDA license) or its antigen-binding fragment, conjugate, or variant. Pembrolizumab is designated CAS Registry No. 1374853-91-4 and is also known as lanizumab, MK-3475, and SCH-900475. Pembrolizumab is an immunoglobulin G4 anti-(human protein PDCD1 (programmed cell death 1)) antibody containing a (human-mouse monoclonal heavy chain) disulfide bond and a human-mouse monoclonal light chain dimer structure. The structure of pembrolizumab can also be described as an immunoglobulin G4 anti-(human programmed cell death 1) antibody; containing a humanized mouse monoclonal [228-L-proline (H10-S>P)]γ4 heavy chain (134-218') disulfide bond and a humanized mouse monoclonal κ light chain dimer (226-226”:229-229”) disulfide bond. The properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO 2008/156712 A1, US Patent No. 8,354,509, and US Patent Application Publications Nos. US2010/0266617 A1, US2013/0108651A1, and US2013/0109843 A2, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of pembrolizumab in various forms of cancer are described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert et al., Lancet, 2014, 384, 1109-17; and Thomas et al., Exp. Opin. Biol. Ther., 2014, 14, 1061-1064. The amino acid sequence of pembrolizumab is illustrated in Table 19. Pembrolizumab includes the following disulfide bridges: 22-96, 22”-96”, 23”-92”’, 23”’-92”’, 134-218”, 134”-218”’, 138”-198”, 138”’-198”’, 147-203”, 147”-203”, 226-226”, 229-229”, 261-321”, 261”-321”, 367-425”, and 367”-425”; and the following glycosylation sites. (N): Asn-297 and Asn-297". Pembrolizumab is an IgG4/κ isoform containing a stable S228P mutation in the Fc region; the insertion of this mutation in the IgG4 hinge region prevents the formation of the half-molecule typically observed in IgG4 antibodies. Pembrolizumab is heteroglycosylated at Asn297 in the Fc domain of each heavy chain, resulting in a molecular weight of approximately 149 kDa for the complete antibody. The major glycoform of pembrolizumab is the fucosylated degalactosyl biantennary (GOF) form.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:168 and the light chain provided in SEQ ID NO:169. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of pembrolizumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:170, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:171 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises _{VH and VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises VH and VL regions} , each at least 97% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:175, SEQ ID NO:176, and SEQ ID NO:177, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of PD-1 approved by a regulatory agency, referencing pembrolizumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab.

Table 19. Amino acid sequences of PD-1 inhibitors associated with pembrolizumab.

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat melanoma. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat melanoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat melanoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat NSCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat small cell lung cancer (SCLC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat SCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat SCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat head and neck squamous cell carcinoma (HNSCC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat HNSCC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HNSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, children are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat MSI-H or dMMR cancer. In some implementations, pediatrics are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat MSI-H or dMMR cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient colorectal cancer (dMMR CRC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat MSI-H or dMMR CRC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat gastric cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat gastric cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat esophageal cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat esophageal cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cervical cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cervical cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat hepatocellular carcinoma (HCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are treated with pembrolizumab at approximately 200 mg every 3 weeks for Merkel cell carcinoma (MCC). In some implementations, adults are treated with pembrolizumab at approximately 400 mg every 6 weeks for MCC. In some implementations, children are treated with pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks for MCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat renal cell carcinoma (RCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks and axitinib 5 mg is administered orally twice daily to treat RCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat endometrial cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks, and lenvatinib 20 mg is administered orally once daily for non-MSI-H or dMMR tumors to treat endometrial cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are given pembrolizumab at approximately 200 mg every 3 weeks to treat TMB-H cancer. In some implementations, adults are given pembrolizumab at approximately 400 mg every 6 weeks to treat TMB-H cancer. In some implementations, children are given pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat TMB-H cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cutaneous squamous cell carcinoma (cSCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat triple-negative breast cancer (TNBC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat TNBC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, if the patient or subject is an adult, i.e., for the treatment of an adult indication, an additional dosing regimen of 400 mg every 6 weeks may be used. In some implementations, pembrolizumab administration may be initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration may be initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 clone J43 (catalog number BE0033-2) and RMP1-14 (catalog number BE0146) (Bio XCell, West Lebanon, New Hampshire, USA). Many commercially available anti-PD-1 antibodies are known to those skilled in the art.

In some embodiments, the PD-1 inhibitor is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publications Nos. 2010/0266617A1, 2013/0108651A1, and 2013/0109843A2, the disclosures of which are incorporated herein by reference. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody described in U.S. Patent Nos. 8,287,856, 8,580,247, and 8,168,757 and U.S. Patent Application Publications Nos. 2009/0028857A1, 2010/0285013A1, 2013/0022600A1, and 2011/0008369A1, the teachings of which are hereby incorporated by reference. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in U.S. Patent No. 8,735,553B1, the disclosure of which is incorporated herein by reference. In some embodiments, the PD-1 inhibitor is pilithumab, also known as CT-011, described in U.S. Patent No. 8,686,119, the disclosure of which is incorporated herein by reference.

In some embodiments, the PD-1 inhibitor may be a small molecule or a peptide or peptide derivative, such as those described in U.S. Patent Nos. 8,907,053, 9,096,642, and 9,044,442, and U.S. Patent Application Publication No. US2015/0087581; 1,2,4-oxadiazole compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2015/0073024; cyclic peptide mimics and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042; cyclic compounds and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042. Those described in US2015/0125491; 1,3,4-oxadiazole and 1,3,4-thiadiazole compounds and derivatives, such as those described in International Patent Application Publication WO 2015/033301; peptide-based compounds and derivatives, such as those described in International Patent Application Publications WO 2015/036927 and WO 2015/04490; or peptide-based macrocyclic compounds and derivatives, such as those described in US Patent Application Publication US2014/0294898; the disclosure of each of these patents is hereby incorporated by reference in its entirety. In some embodiments, the PD-1 inhibitor is cemiplimab, which is available from Regeneron Corporation.

In some embodiments, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors, antagonists, or blockers described in more detail below. Regarding PD-L1 and PD-L2 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor may also refer to the compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

In some embodiments, the compositions, processes, and methods described herein include PD-L1 or PD-L2 inhibitors. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2. In some embodiments, the antibody competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selective for PD-L1 because the concentration at which the compound binds to or interacts with PD-L1 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L2 receptor. In some embodiments, the compound binds to the PD-L1 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L2 receptor.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2 because the concentration at which the compound binds to or interacts with PD-L2 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L1 receptor. In some embodiments, the compound binds to the PD-L2 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L1 receptor.

Unbound by any theory, it is believed that tumor cells express PD-L1, and T cells express PD-1. However, tumor cell expression of PD-L1 is not required for the efficacy of PD-1 or PD-L1 inhibitors or blockers. In some embodiments, tumor cells express PD-L1. In other embodiments, tumor cells do not express PD-L1. In some embodiments, the method may include a combination of PD-1 and PD-L1 antibodies (e.g., the PD-1 and PD-L1 antibodies described herein) with TIL. The combination of PD-1 and PD-L1 antibodies with TIL may be administered simultaneously or sequentially.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that binds to human PD-L1 and/or PD-L2 with a KD of about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, or about 30 pM or less.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that _{bind to human PD-L1 and/or PD-L2 at a k-association rate of} about ^7.5 × 10⁵ ¹ /M·s or faster, at about 8 × 10⁵ 1/M·s or _faster , at about 8.5 × 10⁵ ¹ /M·s or faster, at about 9 × ^10⁵ 1/M·s or _faster , at about 9.5 × ^10⁵ 1/M·s or _faster , or at about 1 × ^10⁶ 1/M·s or faster. 1/M·s or faster k- _{associations bind} to human PD-L1 and/or PD-L2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that bind to human PD-L1 or PD-L2 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k-dissociation rate of about 2.1 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.3 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, to human PD-1 with _{a k-dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, and to human PD-1 with a k _{-dissociation rate} of about 2.6 × ^{10⁻⁵ 1/s} or slower. It binds to human PD-1 at a k _{dissociation rate} of 1/s or slower, to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 2.7 × ^10⁻⁵ 1/s or slower, or to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, with an IC50 of about 9 nM or less, with an IC50 of about 8 nM or less, with an IC50 of about 7 nM or less, and with an IC50 of about 6 nM or less. It can either inhibit the binding of human PD-L1 or human PD-L2 to human PD-1, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower, or block human PD-1 or block the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-L1 inhibitor is durvalumab, also known as MEDI4736 (available from Medimmune, LLC (a subsidiary of AstraZeneca plc) in Gaithersburg, Maryland), or its antigen-binding fragment, conjugate, or variant. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013/0034559, the disclosure of which is incorporated herein by reference. The clinical efficacy of durvalumab has been described in Page et al., Ann. Rev. Med., 2014, 65, 185-202; Brahmer et al., J. Clin. Oncol. 2014, 32, 5s (Supplement, Abstract 8021); and McDermott et al., Cancer Treatment Rev., 2014, 40, 1056-64. The preparation and characterization of durvalumab are described in U.S. Patent No. 8,779,108, the disclosure of which is incorporated herein by reference. The amino acid sequence of durvalumab is set forth in Table 20. The durvalumab monoclonal antibody comprises disulfide bonds at 22-96, 22”-96”, 23”-89”’, 23”’-89”’, 135”-195”’, 135”’-195”’, 148”-204”, 148”-204”, 215”-224”, 215”’-224”, 230-230”, 233-233”, 265-325”, 265”-325”, 371-429” and 371”-429”’; and N-glycosylation sites at Asn-301” and Asn-301”.

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:178 and the light chain provided in SEQ ID NO:179. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of the durvalumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:180, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:181 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises VH and _{VL regions} , each at least 99% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:182, SEQ ID NO:183, and SEQ ID NO:184, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:185, SEQ ID NO:186, and SEQ ID NO:187, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of anti-PD-L1 approved by a regulatory agency, referencing durvalumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab.

Table 20. Amino acid sequences of PD-L1 inhibitors associated with durvalumab.

In some embodiments, the PD-L1 inhibitor is avelumab, also known as MSB0010718C (available from MerckKGaA/EMD Serono) or its antigen-binding fragment, conjugate, or variant. The preparation and characterization of avelumab are described in U.S. Patent Application Publication No. US2014/0341917 A1, the disclosure of which is incorporated herein by reference. The amino acid sequence of avelumab is set forth in Table 21. Avirumab contains heavy chain intra-chain disulfide bonds (C23-C104) at 22-96, 147-203, 264-324, 370-428, 22”-96”, 147”-203”, 264”-324”, and 370”-428”; and light chain intra-chain disulfide bonds (C23-C104) at 22’-90”, 138’-197”, 22”’-90”’, and 138”’-197”’; 223- Intra-heavy disulfide bonds between the heavy and light chains at 215' and 223"-215"' (h 5-CL 126); intra-heavy disulfide bonds between the heavy and light chains at 229"-229" and 232"-232" (h11, h 14); N-glycosylation sites at 300" and 300" (H CH2 N84.4); fucoidan-like CHO polysaccharides of the fucoidan complex; and H CHS K2 C-terminal lysine cleavage at 450" and 450".

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:188 and the light chain provided in SEQ ID NO:189. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of avirumab. In some embodiments, the heavy chain variable region (VH) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:190, and the light chain variable region (VL) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:191 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:195, SEQ ID NO:196, and SEQ ID NO:197, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of an anti-PD-L1 antibody approved by a pharmaceutical regulatory agency, referencing avirumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. The anti-PD-L1 antibody may be authorized by a pharmaceutical regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is avelumab.

Table 21. Amino acid sequences of PD-L1 inhibitors associated with avirumab.

In some embodiments, the PD-L1 inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (available from Genentech (a subsidiary of Roche Holding AG) in Basel, Switzerland, under the TECENTRIQ license), or an antigen-binding fragment, conjugate, or variant thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent Application Publications Nos. 2010/0203056A1, 2013/0045200A1, 2013/0045201A1, 2013/0045202A1, or 2014/0065135A1, the disclosure of which is specifically incorporated herein by reference. The preparation and characterization of atezolizumab are described in U.S. Patent No. 8,217,149, the disclosure of which is incorporated herein by reference. The amino acid sequence of atezolizumab is set forth in Table 22. Atezolizumab has intrachain disulfide bonds (C23-C104) at 22-96, 145-201, 262-322, 368-426, 22”-96”, 145”-201”, 262”-322”, and 368”-426”; and at 23”-88”, 134”-194”, 23”’-88”’, and 134”’-194”’. Intra-light chain disulfide bonds (C23-C104); intra-heavy chain disulfide bonds at 221-214' and 221”-214”' (h 5-CL 126); intra-heavy chain disulfide bonds at 227-227” and 230-230” (h 11, h 14); and N-glycosylation sites at 298 and 298' (H CH2 N84.4>A).

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:198 and the light chain provided in SEQ ID NO:199. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of atezolizumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:200, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:201 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, _respectively . In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:204, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:205, SEQ ID NO:206, and SEQ ID NO:207, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the anti-PD-L1 antibody is a biosimilar monoclonal antibody of atezolizumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab.

Table 22. Amino acid sequences of PD-L1 inhibitors associated with atezolizumab.

In some embodiments, the PD-L1 inhibitor includes those antibodies described in U.S. Patent Application Publication No. 2014/0341917 A1, the disclosure of which is incorporated herein by reference. In other embodiments, an antibody competing with any of these antibodies for binding to PD-L1 is also included. In some embodiments, the anti-PD-L1 antibody is MDX-1105, also known as BMS-935559, disclosed in U.S. Patent No. 7,943,743, the disclosure of which is incorporated herein by reference. In some embodiments, the anti-PD-L1 antibody is selected from the anti-PD-L1 antibody disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference.

In some embodiments, the PD-L1 inhibitor is a commercially available monoclonal antibody, such as INVIVOMAB anti-m-PD-L1 clone 10F.9G2 (catalog number BE0101, Bio X Cell, West Lebanon, New Hampshire, USA). In some embodiments, the anti-PD-L1 antibody is a commercially available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). Many commercially available anti-PD-L1 antibodies are known to those skilled in the art.

In some implementations, the PD-L2 inhibitor is a commercially available monoclonal antibody, such as BIOLEGEND24F.10C12 mouse IgG2a, κ isotype (catalog number 329602, Biolegend, San Diego, California), SIGMA anti-PD-L2 antibody (catalog number SAB3500395, Sigma-Aldrich, St. Louis, Missouri), or other commercially available anti-PD-L2 antibodies known to those skilled in the art.

2. Combination with CTLA-4 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, including TIL and one or more CTLA-4 inhibitors.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily and is expressed on the surface of helper T cells. CTLA-4 is a negative regulator of CD28-dependent T cell activation and acts as a checkpoint for adaptive immune responses. Similar to the T cell costimulatory protein CD28, CTLA-4 binds to antigens and presents CD80 and CD86 on cells. CTLA-4 delivers inhibitory signals to T cells, while CD28 delivers stimulatory signals. Human antibodies against human CTLA-4 have been described as immunomodulators for many disease conditions, such as for the treatment or prevention of viral and bacterial infections and for the treatment of cancer (WO 01/14424 and WO 00/37504). Several fully human anti-human CTLA-4 monoclonal antibodies (mAbs) have been investigated in clinical trials for the treatment of various types of solid tumors, including (but not limited to) ipilimumab (MDX-010) and trimemumab (CP-675,206).

In some embodiments, the CTLA-4 inhibitor may be any CTLA-4 inhibitor or CTLA-4 blocker known in the art. In particular, it is one of the CTLA-4 inhibitors or blockers described in more detail below. Regarding CTLA-4 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or bioanalyte. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor may also refer to a small molecule compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

CTLA-4 inhibitors suitable for the methods of the present invention include (but are not limited to) anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (ipilimumab), trimemumab, anti-CD28 antibodies, anti-CTLA-4 adenettin, anti-CTLA-4 domain antibodies, and single-chain anti-CTLs. The following are listed: A-4 fragment, heavy chain anti-CTLA-4 fragment, light chain anti-CTLA-4 fragment, CTLA-4 inhibitor activating the co-stimulatory pathway, antibody disclosed in PCT Publication No. WO 2001/014424, antibody disclosed in PCT Publication No. WO 2004/035607, antibody disclosed in U.S. Publication No. 2005/0201994, and antibody disclosed in European Patent No. EP 1212422 B1, the disclosures of each of which are incorporated herein by reference. Other CTLA-4 antibodies are described in U.S. Patents 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publications WO 01/14424 and WO 00/37504; and U.S. Publications 2002/0039581 and 2002/086014, the contents of each of which are incorporated herein by reference. Other anti-CTLA-4 antibodies that may be used in the methods of this invention include, for example, antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (Antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998); and U.S. Patents 5,977,318, 6,682,736, 7,109,003 and 7,132,281, the disclosure of each of which is incorporated herein by reference.

Other CTLA-4 inhibitors include (but are not limited to) the following: those that, by means of activation, disrupt the ability of CD28 antigen to bind to its homologous ligand, inhibit the ability of CTLA-4 to bind to its homologous ligand, enhance T cell responses via the co-stimulatory pathway, disrupt the ability of B7 to bind to CD28 and/or CTLA-4, disrupt the ability of B7 to activate the co-stimulatory pathway, disrupt the ability of CD80 to bind to CD28 and/or CTLA-4, disrupt the ability of CD80 to activate the co-stimulatory pathway, disrupt the ability of CD86 to bind to CD28 and/or CTLA-4, disrupt the ability of CD86 to activate the co-stimulatory pathway, and any inhibitor that disrupts the co-stimulatory pathway. This must include: small molecule inhibitors of CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antibodies against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antisense molecules against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; adenettin against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; RNAi inhibitors (single-stranded and double-stranded) against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; and other CTLA-4 inhibitors.

In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 at a K d , which is about 10  -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, for example, between 10 -13 M and 10 -16 M, or within any range with any two of the foregoing values as endpoints. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is no more than 10 times that of the Kd of ipilimumab. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is substantially the same as or less than that of ipilimumab (e.g., up to 10 times or up to 100 times lower). In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is no more than 10-fold higher than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively. In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is approximately the same as or less than (e.g., up to 10-fold or up to 100-fold lower) than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively.

In some embodiments, a CTLA-4 inhibitor is used in an amount sufficient to inhibit CTLA-4 expression and/or reduce the biological activity of CTLA-4 by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100%. In some embodiments, a CTLA-4 pathway inhibitor is used in an amount sufficient to reduce the biological activity of CTLA-4 by reducing the binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100% relative to a suitable control. In the context of assessing or quantifying the effect of an agent of interest, a suitable control is typically a biological system (e.g., cells or a subject) that has not been exposed to the agent of interest (e.g., a CTLA-4 pathway inhibitor) or has been treated with the agent (or has been exposed to or treated with a negligible amount). In some embodiments, the biological system may serve as its own control, for example, by assessing the biological system before exposure to or treatment with the agent and comparing its status after the start or end of exposure or treatment. In some embodiments, a historical control may be used.

In some embodiments, the CTLA-4 inhibitor is ipilimumab (available from Bristol-Myers Squibb at Yervoy) or its biosimilars, antigen-binding fragments, conjugates, or variants. As known in the art, ipilimumab refers to an anti-CTLA-4 antibody, a fully human IgG 1κ antibody derived from transgenic mice having human genes encoding both heavy and light chains to produce a functional human lineage. Ipilimumab is also mentioned in its CAS Registry No. 477202-00-9 and in PCT Publication No. WO 01/14424, which is incorporated herein by reference in its entirety for all purposes. It is disclosed in the form of Antibody 10DI. Specifically, ipilimumab contains a light chain variable region and a heavy chain variable region (having a light chain variable region comprising SEQ ID NO: 211 and a heavy chain variable region comprising SEQ ID NO: 210). Pharmaceutical compositions of ipilimumab comprise all pharmaceutically acceptable compositions containing ipilimumab and one or more diluents, mediators, or excipients. Examples of pharmaceutical compositions containing ipilimumab are described in International Patent Application Publication No. WO 2007/67959. Ipilimumab can be administered intravenously (IV).

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:208 and the light chain provided in SEQ ID NO:209. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of ipilimumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:210, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:211 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:215, SEQ ID NO:216, and SEQ ID NO:217, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to ipilimumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of ipilimumab is illustrated in Table 23. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab.

Table 23. Amino acid sequence of ipilimumab.

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, ipilimumab administration may be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, ipilimumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab is administered at approximately mg/kg every 3 weeks for up to 4 consecutive doses to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered as adjuvant therapy for melanoma. In some implementations, ipilimumab is administered at approximately 10 mg/kg every 3 weeks for four doses, followed by 10 mg/kg every 12 weeks for up to 3 years as adjuvant therapy for melanoma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat advanced renal cell carcinoma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 3 weeks, followed by nivolumab at 3 mg/kg on the same day, for a total of 4 doses to treat advanced renal cell carcinoma. In some implementations, after completing the combined 4-dose regimen, nivolumab can be administered as a single agent against advanced renal cell carcinoma and/or renal cell carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, ipilimumab is administered intravenously at approximately 1 mg/kg every 30 minutes over 3 weeks, followed immediately by nivolumab intravenously at 3 mg/kg every 30 minutes on the same day, for a total of 4 doses to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, after completing 4 doses of the composition, nivolumab is administered as a single agent for microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, as recommended according to standard dosing regimens. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also begin 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat hepatocellular carcinoma. In some implementations, ipilimumab is administered intravenously at approximately 3 mg/kg every 3 weeks over 30 minutes, followed immediately by nivolumab intravenously at 1 mg/kg over 30 minutes on the same day, for a total of four doses to treat hepatocellular carcinoma. In some implementations, after completing the combined four-dose regimen, nivolumab is administered as a single agent against hepatocellular carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, followed by nivolumab at 3 mg/kg every 2 weeks to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, plus 360 mg nivolumab every 3 weeks for 2 cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat malignant pleural mesothelioma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks and nivolumab at 360 mg every 3 weeks to treat malignant pleural mesothelioma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

Trimelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has CAS number 745013-59-6. Trimelimumab is disclosed in U.S. Patent No. 6,682,736 (incorporated herein by reference) as antibody 11.2.1. The amino acid sequences of the heavy and light chains of trimemumab are described in SEQ IND NO: 218 and 219, respectively. Trimelimumab has been studied in clinical trials for the treatment of various tumors, including melanoma and breast cancer; wherein it is administered intravenously in single or multiple doses at a dose range between 0.01 and 15 mg/kg every 4 or 12 weeks. In the embodiments provided by the present invention, trimemumab is administered topically, particularly intradermally or subcutaneously. The effective amount of trimemumab administered intradermally or subcutaneously is typically in the range of 5-200 mg/dose per person. In some implementations, the effective dose of trimemumab is in the range of 10-150 mg/dose per person. In some specific implementations, the effective dose of trimemumab is approximately 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175 or 200 mg/dose per person.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:218 and the light chain provided in SEQ ID NO:219. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of trimexumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:220, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:221 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in _SEQ ID NO:220 and _SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:225, SEQ ID NO:226, and SEQ ID NO:227, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a biosimilar monoclonal antibody of anti-CTLA-4 approved by a regulatory agency, referencing trimemumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of trimemumab is illustrated in Table 24. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab.

Table 24. Amino acid sequence of trimemumab.

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, wherein trimemumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, trimemumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, trimemumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is zeflimab from Agenus or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Zeflimab is a fully human monoclonal antibody. Zeflimab is designated Chemical Abstracts Service (CAS) Registry No. 2148321-69-9 and is also known as AGEN1884. The preparation and characterization of zeflimab are described in U.S. Patent No. 10,144,779 and U.S. Patent Application Publication No. US2020/0024350A1, the disclosures of which are incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:228 and the light chain provided in SEQ ID NO:229. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of zeflimab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:230, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:231 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:231, SEQ ID NO:233, and SEQ ID NO:234, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:235, SEQ ID NO:236, and SEQ ID NO:237, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to zeflimab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of zeflimab is illustrated in Table 25. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab.

Table 25. Amino acid sequence of zefrizinumab.

Other examples of anti-CTLA-4 antibodies include (but are not limited to): AAGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659, and ADG116, which are known to those skilled in the art.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent disclosures: US2019/0048096 A1; US2020/0223907; US2019/0201334; US2019/0201334; US2005/0201994; EP 1212422B1; WO 2018/204760; WO 2018/204760; WO 2001/014424; WO2 WO 2004/035607; WO 2003/086459; WO 2012/120125; WO 2000/037504; WO 2009/100140; WO2006/09649; WO2005092380; WO 2007/123737; WO 2006/029219; WO 2010/0979597; WO2006/12168; and WO1997020574, each of which is incorporated herein by reference. Other CTLA-4 antibodies are described in the following: U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227 and 6,984,720; PCT Publications Nos. WO 01/14424 and WO 00/37504; and U.S. Publications Nos. 2002/0039581 and 2002/086014; and/or U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281, each of which is incorporated herein by reference. In some embodiments, the anti-CTLA-4 antibody is, for example, one of the antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 10067-10071 (1998); Camacho et al., J. Clin. Oncol., 2004, 22, 145 (Abstract No. 2505 (2004) (Antibody CP-675206); or Mokyr et al., Cancer Res., 1998, 58, 5301-5304 (1998), each of which is incorporated herein by reference.

In some implementations, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996/040915 (incorporated herein by reference).

In some implementations, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, anti-CTLA-4 RNAi molecules may be in the form described in: PCT Publications WO 1999/032619 and WO 2001/029058; U.S. Publications 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, and 2005/01009. Patent Nos. 13, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443; and/or U.S. Patent Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in European Patent No. 1309726 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in U.S. Patent Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In some embodiments, the CTLA-4 inhibitor is an aptamer described in International Patent Application Publication No. WO2004/081021 (incorporated herein by reference).

In other embodiments, the anti-CTLA-4 RNAi molecule of the present invention is the RNA molecule described in U.S. Patent Nos. 5,898,031, 6,107,094, 7,432,249 and 7,432,250 and European Application No. EP 0928290 (incorporated herein by reference).

3. Preconditioning of lymphocyte depletion in patients

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the present disclosure. In some embodiments, the present invention includes a TIL population for treating cancer in patients pretreated with non-myeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/ ^m² /day for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion (day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 (aldeleukin, available from PROLEUKIN) every 8 hours at 720,000 IU/kg to achieve physiological tolerance. In some implementations, TIL groups are used in combination with IL-2 to treat cancer, wherein IL-2 is administered after the TIL group.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system ('cytokine pool') prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as "immunosuppressive modulation") in a patient prior to the introduction of the TIL of the present invention.

Generally, lymphocyte depletion is achieved by administration of fludarabine or cyclophosphamide (the active form of which is called equiphosphamide) and combinations thereof. Such methods are described in Gassner et al., Cancer Immunol. Immunother. 2011, 60, 75-85; Muranski et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-5239; and Dudley et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL. In some embodiments, fludarabine is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, fludarabine is administered at doses of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2–7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4–5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4–5 days.

In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 1 μg/mL. In some embodiments, cyclophosphamide is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, cyclophosphamide is administered at doses of 100 mg/m²/day, 150 mg/m²/day, 175 mg/m²/day, 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day, 275 mg/m²/day, or 300 mg/m²/day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.e., i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2–7 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4-5 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4 days.

In some implementations, lymphocyte depletion is achieved by administering fludarabine and cyclophosphamide together to the patient. In some implementations, fludarabine is administered intravenously at 25 mg/m²/day for 4 days, and cyclophosphamide is administered intravenously at 250 mg/m²/day.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days and fludarabine at a dose of 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered for the first two days and lymphocyte depletion is achieved over a total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 15 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by fludarabine at a dose of 25 mg/m²/day for three days.

In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, mesna is administered at a dose of 15 mg/kg. In some embodiments, mesna is infused, and if continuous infusion is initiated over 24 hours, with the respective cyclophosphamide dose starting, mesna may be infused with cyclophosphamide over approximately 2 hours (days -5 and/or -4), and then infused at a rate of 3 mg/kg/hour for the remaining 22 hours.

In some implementations, lymphocyte depletion includes the following steps: starting the day after administering the third TIL group to the patient, treating the patient with an IL-2 regimen.

In some implementations, lymphocyte depletion includes the following steps: starting on the day the third TIL group is administered to the patient, the patient is treated with an IL-2 regimen.

In some embodiments, lymphocyte depletion comprises a 5-day preconditioning treatment. In some embodiments, the days are indicated as day-5 to day-1, or day 0 to day 4. In some embodiments, the regimen comprises cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises 60 mg/kg intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, the regimen also comprises fludarabine. In some embodiments, the regimen also comprises intravenous fludarabine. In some embodiments, the regimen also comprises 25 mg/ ^m² intravenous fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine at a dose of 25 mg/ ^m² on days -5 and ^-1 (i.e., days 0 to 4).

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for one day.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 26.

Table 26. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 27.

Table 27. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 28.

Table 28. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 29.

Table 29. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day			X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 30.

Table 30. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 31.

Table 31. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 32.

Table 32. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 33.

Table 33. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day			X	X	X
TIL infusion						X

In some embodiments, the TIL infusion used in conjunction with the above-described myeloablative lymphocyte depletion regimens may be any TIL composition described herein, as well as an IL-2 addition regimen and administration of combination therapies as described herein (e.g., PD-1 and PD-L1 inhibitors).

4. IL-2 scheme

In some implementations, the IL-2 regimen includes a high-dose IL-2 regimen comprising alephrine or a biosimilar or variant thereof, administered intravenously starting the day after the effective fraction of treatment in the therapeutic TIL population. Alephrine or a biosimilar or variant thereof is administered as a 15-minute bolus intravenous infusion every eight hours at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient weight) until tolerated, for a maximum of 14 doses. After a 9-day rest period, this course can be repeated for another 14 doses, for a maximum total of 28 doses. In some implementations, IL-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In some implementations, IL-2 is administered at a maximum dose of up to 6 doses.

In some implementations, the IL-2 regimen comprises a tapering IL-2 regimen. Tapering IL-2 regimens have been described in O'Day et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In some implementations, tapering IL-2 therapy comprises an intravenous administration of 18 × ^10⁶ IU/ ^m² over 6 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 12 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 24 hours, followed by an intravenous administration of 4.5 × ^10⁶ IU/ ^m² of adeleukin or a biosimilar or variant thereof over 72 hours. This treatment cycle may be repeated every 28 days for a maximum of four cycles. In some implementations, the decreasing IL-2 regimen includes 18,000,000 IU/ ^m² on day 1, 9,000,000 IU/ ^m² on day 2, and 4,500,000 IU/ ^m² on days 3 and 4.

In some implementations, the IL-2 regimen includes a low-dose IL-2 regimen. Any low-dose IL-2 regimen known in the art may be used, including those described in Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann et al., Lancet Diabetes Endocrinol. 2013, 1, 295-305; and Rosenzwaig et al., Ann. Rheum. Dis. 2019, 78, 209-217, the disclosures of which are incorporated herein by reference. In some implementations, a low-dose IL-2 regimen comprises 18 × ^10⁶ IU/ ^m² of interleukin or its biosimilar or variant every 24 hours, administered as a continuous infusion for 5 days; followed by a 2–6 day period without IL-2 therapy; optionally followed by intravenous administration of interleukin or its biosimilar or variant at 18 × ^10⁶ IU/ ^m² every 24 hours for another 5 days; optionally followed by a 3-week period without IL-2 therapy, after which other cycles may be administered.

In some implementations, IL-2 is administered at a maximum dose of up to 6 doses. In some implementations, high-dose IL-2 regimens are modified for pediatric use. In some implementations, interleukin is administered at a dose of 600,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 400,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 300,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 200,000 IU/kg every 8-12 hours, for a maximum of 6 doses. In some implementations, interleukin is administered at a dose of 100,000 IU/kg every 8-12 hours, for a maximum of 6 doses.

In some embodiments, the IL-2 regimen comprises administering PEGylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bepedipine interleukin or a fragment, variant, or bioanalyte thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen comprises administering THOR-707 or a fragment, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen includes administration of Nevorin Alpha or a fragment, variant, or biosimilar thereof following TIL administration. In some implementations, Nevorin Alpha is administered to the patient at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 protocol includes administering an IL-2 fragment grafted onto the antibody backbone. In some embodiments, the IL-2 protocol includes administering an antibody cytokine graft protein that binds to a low-affinity IL-2 receptor. In some embodiments, the antibody cytokine graft protein includes a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof grafted into the CDR of _VH or _VL , wherein the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof, which is transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody or a fragment thereof, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days, wherein the antibody comprises a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

In some implementations, the serum half-life of the antibody cytokine transplanted protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to interleukin or comparable molecules).

In some embodiments, the TIL infusion used in conjunction with the aforementioned embodiments of the myeloablative lymphocyte depletion regimen may be any TIL composition described herein and may also include MIL and PBL infusions in lieu of TIL infusion, as well as the addition of an IL-2 regimen and administration of combination therapies as described herein (e.g., PD-1 and/or PD-L1 inhibitors and/or CTLA-4 inhibitors).

Implementation Plan

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating the fourth TIL population takes about 9 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 bead agonist or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about one day.

In some implementations, the process of cultivating the fourth TIL population takes about 2 days.

In some implementations, the process of cultivating a fourth TIL population takes about 3 days.

In some implementations, the process of cultivating the fourth TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing a third TIL population takes about 8 days.

In some implementations, the process of culturing a third TIL population takes approximately 9 days.

In some implementations, the process of culturing the third TIL population takes about 8-9 days.

In some implementations, the process of cultivating a third TIL population takes about 10 days.

In some implementations, the process of culturing a third TIL population takes approximately 8-10 days.

In some implementations, the process of culturing a third TIL population takes approximately 11 days.

In some implementations, the process of culturing a third TIL population takes approximately 12 days.

In some implementations, the process of cultivating a third TIL population takes approximately 13 days.

In some implementations, the process of culturing a third TIL population takes approximately 14 days.

In some implementations, the process of cultivating a third TIL population takes about 15 days.

In some implementations, the step of culturing a third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about one day.

In some implementations, the process of cultivating a third TIL population takes about 2 days.

In some implementations, the process of cultivating a third TIL population takes about 3 days.

In some implementations, the process of cultivating a third TIL population takes about 4 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) Culture the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) A fourth population of TILs was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately nine days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a third cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a fourth culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the step of culturing a third or fourth TIL population takes about one day.

In some implementations, the process of culturing a third or fourth TIL population takes about 2 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 3 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 4 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 5 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 6 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, during the step of culturing a first TIL population in a first culture medium, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in the first culture medium.

In some embodiments, during the step of culturing a second TIL population in a second culture medium, the second culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in a second culture medium.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of a first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the initiation of the first amplification takes approximately one day.

In some implementations, the initiation of the first amplification takes approximately 2 days.

In some implementations, the initiation of the first amplification takes approximately 3 days.

In some implementations, the initiation of the first amplification takes approximately 4 days.

In some implementations, the initiation of the first amplification takes approximately 5 days.

In some implementations, the initiation of the first amplification takes approximately 6 days.

In some implementations, the initiation of the first amplification takes approximately 7 days.

In some implementations, the initiation of the first amplification takes approximately 8 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the rapid second amplification takes approximately 9 days.

In some implementations, the rapid second amplification takes approximately 10 days.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, all steps are completed within a period of approximately 16 days.

In some implementations, all steps are completed within a period of approximately 17 days.

In some implementations, all steps are completed within a period of approximately 18 days.

In some implementations, all steps are completed within a period of approximately 18-22 days.

In some implementations, all steps are completed within a period of approximately 19 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 21 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, the first culture medium contains APC.

In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

In some embodiments, the first culture medium contains OKT-3.

In some implementations, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies during the initial amplification step.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digesting tumor tissue samples in an enzyme medium to produce tumor digests;

(c) The tumor digest is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (f), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the first amplification takes about 3 days.

In some implementations, the first amplification takes about 4 days.

In some implementations, the first amplification takes about 5 days.

In some implementations, the first amplification takes about 6 days.

In some implementations, the first amplification takes about 7 days.

In some implementations, the first amplification takes about 8 days.

In some implementations, the first amplification takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes about 5 days.

In some implementations, the second amplification takes about 6 days.

In some implementations, the second amplification takes about 7 days.

In some implementations, the second amplification takes approximately 8 days.

In some implementations, the second amplification takes approximately 9 days.

In some implementations, the second amplification takes about 8-9 days.

In some implementations, the second amplification takes about 10 days.

In some implementations, the second amplification takes about 8-10 days.

In some implementations, the second amplification takes approximately 11 days.

In some implementations, the second amplification takes approximately 12 days.

In some implementations, the second amplification takes approximately 13 days.

In some implementations, the second amplification takes approximately 14 days.

In some implementations, the second amplification takes about 15 days.

In some implementations, the second amplification is performed through the following steps:

(i) The first period of culturing the fourth TIL population in the second culture medium for about 5 days.

(ii) The culture from step (i) is subdivided into multiple subcultures, each of which is transferred to a separate sealed container providing a third breathable surface and cultured for a second period of approximately 4 or 5 days in a third medium containing IL-2, wherein the transition from step (i) to step (ii) is performed without opening the system, and

(iii) Multiple subcultures are merged to produce a fifth TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, all steps are completed within a period of approximately 25 days.

In some implementations, all steps are completed within a period of approximately 26 days.

In some implementations, all steps are completed within a period of approximately 27 days.

In some implementations, all steps are completed within a period of approximately 28 days.

In some implementations, all steps are completed within a period of approximately 29 days.

In some implementations, all steps are completed within a period of approximately 30 days.

In some implementations, all steps are completed within a period of approximately 31 days.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Digesting a first TIL population from tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a patient or subject in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, this document provides expanded numbers of tumor-infiltrating lymphocytes (TILs) or therapeutic TIL populations produced by the methods described herein.

In some embodiments, this document provides pharmaceutical compositions comprising an amplified number of TILs or a therapeutic population of TILs as described herein and a pharmaceutically acceptable carrier.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of an expanded number of TILs or a therapeutically active population of TILs as described herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor excised from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor digest to a closed system and performing a first amplification to produce a second TIL population by culturing the first TIL population in a first cell culture medium containing IL-2, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting PD-1 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CISH expression.

In some implementations, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting LAG-3 expression and a TALEN system for inhibiting TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is approximately 2.3 × 10¹⁰ to approximately 13.7 × 10¹⁰ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of TIL cells in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the administration of TIL cells to the patient in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Example

The embodiments covered herein are now described with reference to the following examples. These examples are provided for illustrative purposes only and this disclosure should in no way be construed as limiting to these examples, but rather as covering any and all variations that become apparent from the teachings provided herein.

Example 1: Preparation of culture medium for pre-REP and REP processes

This embodiment describes a procedure for preparing a tissue culture medium suitable for cultures involving tumor-infiltrating lymphocytes (TILs) derived from various solid tumors. This culture medium can be used to prepare any of the TILs described in this application and other embodiments.

Prepare CM1 . Remove the following reagents from the freezer and warm them in a 37°C water bath: (RPMI 1640, human AB serum, 200 mM L-glutamine). Prepare CM1 medium by adding each component to the top of a 0.2 μm filter unit suitable for the desired filtration volume, according to Table 34 below. Store at 4°C.

Table 34. Preparation of CM1

On the day of use, preheat the required amount of CM1 in a 37°C water bath and add 6000 IU/mL IL-2.

Additional information may be added as needed, based on Table 35.

Table 35. Additional information for CM1 as needed.

Preparation of CM2

Remove the prepared CM1 from the refrigerator or prepare fresh CM1. Remove AIM- from the refrigerator and prepare the required amount of CM2 by mixing the prepared CM1 with an equal volume of AIM- in a sterile culture medium bottle. On the day of use, add 3000 IU/mL IL-2 to the CM2 culture medium. On the day of use, prepare a sufficient amount of CM2 with 3000 IU/mL IL-2. Label the CM2 culture medium bottle with the name, the initials of the preparer's name, the filtration/preparation date, and the two-week expiry date, and store at 4°C until needed for tissue culture.

Preparation of CM3

Prepare CM3 on the day it is needed. CM3 is the same as AIM-medium, but supplemented with 3000 IU/mL IL-2 on the day of use. Prepare the amount of CM3 needed for the experiment by directly adding the IL-2 stock solution to the AIM-V bottle or bag. Mix thoroughly by gentle shaking. Immediately after adding to AIM-V, label the bottle “3000 IU/mL IL-2”. If there is excess CM3, store it in a bottle at 4°C, labeling it with the medium name, the preparer's initials, the date of preparation, and its expiration date (7 days after preparation). After storing at 4°C for 7 days, discard the medium supplemented with IL-2.

Preparation of CM4

CM4 is the same as CM3, but with an additional 2 mM GlutaMAX ^™ (final concentration). Add 10 mL of 200 mM GlutaMAX ^™ per 1 L of CM3. Prepare the required amount of CM4 by directly adding IL-2 and GlutaMAX ^™ stock solution to the AIM-V bottle or bag. Mix thoroughly with gentle shaking. Immediately after adding to the AIM-V, label the bottle “3000 IL/mL IL-2 and GlutaMAX”. If excess CM4 exists, store it in the bottle at 4°C, labeling it with the culture medium name, “GlutaMAX”, and its expiration date (7 days after preparation). Discard the culture medium supplemented with IL-2 after storage at 4°C for more than 7 days.

Example 2: Use of a mixture of IL-2, IL-15 and IL-21 cytokines

This embodiment describes the use of IL-2, IL-15, and IL-21 cytokines, which act as additional T cell growth factors, in combination with the TIL process of any embodiment herein.

Using the procedures described herein, at the start of culture, TILs were observed to grow from tumors in one experimental group in the presence of IL-2 and in another group in the presence of a combination of IL-2, IL-15, and IL-21, which substituted for IL-2. At the completion of pre-REP, the amplification, phenotype, function (CD107a+ and IFN-γ), and TCR Vβ lineage of the cultures were assessed. IL-15 and IL-21 are described elsewhere in this paper and in Santegoets et al., J. Transl. Med., 2013, 11, 37.

The results showed enhanced TIL amplification (>20%) in CD4 ⁺ and CD8 ⁺ cells under IL-2, IL-15, and IL-21 treatments compared to IL-2-only conditions. Significant skewness towards a CD8 ⁺ population with a skewed TCR Vβ lineage was observed in TILs obtained from IL-2, IL-15, and IL-21-treated cultures compared to IL-2-only cultures. IFN-γ and CD107a levels were increased in TILs treated with IL-2, IL-15, and IL-21 compared to IL-2-only TILs.

Example 3: Quantification of individual batches of γ-irradiated peripheral mononuclear cells

This embodiment describes a simplified procedure for quantifying individual batches of γ-irradiated peripheral monocytes (PBMCs, also known as monocytes or MNCs) used as allogeneic feeder cells in the exemplary methods described herein.

Each batch of irradiated MNC feeder cells was prepared from individual donors. Each batch or donor was individually screened based on its ability to amplify TILs in the REP in the presence of purified anti-CD3 (clonal OKT3) antibody and interleukin-2 (IL-2). Furthermore, each batch of feeder cells was tested without the addition of TILs to verify that the received gamma irradiation dose was sufficient to prevent replication.

TIL regeneration requires γ-irradiated, growth-arrested MNC feeder cells. Membrane receptors on the feeder MNCs bind to anti-CD3 (clonal OKT3) antibodies and cross-link with TILs in the REP culture flask, stimulating TIL expansion. Feeder cell batches are prepared by leukocyte removal from whole blood obtained from individual donors. The leukocyte removal product is centrifuged, washed, irradiated, and cryopreserved under GMP conditions on a Ficoll-Hypaque apparatus.

Importantly, live feeder cells should not be infused into patients receiving TIL therapy, as this can cause graft-versus-host disease (GVHD). Consequently, feeder cell growth is inhibited by gamma irradiation, leading to double-strand DNA breaks and loss of MNC cell viability upon reculture.

Feeder cell batches were evaluated according to two criteria: (1) their ability to amplify TILs by >100-fold in co-culture, and (2) their insufficient replication capacity.

Feeder cell batches were tested in a mini-REP format using two primary pre-REP TIL lines grown in vertical T25 tissue culture flasks. Feeder cell batches were tested against two distinct TIL lines, each with a unique ability to proliferate in response to activation within the REP. As a control, a number of previously demonstrated irradiated MNC feeder cells meeting the above criteria were processed with the test batches.

A stock solution of the same pre-REP TIL line is available to test all conditions and all feeder cell batches, ensuring that all batches tested in a single experiment undergo equivalent testing.

For each batch of feeder cells tested, a total of six T25 culture flasks were used: pre-REP TIL line #1 (2 flasks); pre-REP TIL line #2 (2 flasks); and feeder cell control (2 flasks). Flasks containing TIL lines #1 and #2 were used to evaluate the feeder cell batch's ability to amplify TILs. The feeder cell control flasks were used to evaluate insufficient replication capacity in the feeder cell batch.

A. Experimental Protocol

On day -2/3, thaw the TIL culture . Prepare CM2 medium and warm it in a 37°C water bath. Prepare 40 mL of CM2 supplemented with 3000 IU/mL IL-2. Keep warm until used. Place 20 mL of pre-warmed CM2 (without IL-2) into each of two 50 mL conical tubes labeled with the name of the TIL culture used. Remove the two designated pre-REP TIL cultures from the LN2 reservoir and transfer the vials to the tissue culture room. Thaw the vials by placing them in a sealed zip-lock storage bag in a 37°C water bath until a small amount of ice remains.

Using a sterile pipette, immediately transfer the contents of each vial to 20 mL of CM2 in a prepared, labeled 50 mL conical tube. Make up to 40 mL with IL-2-free CM2 to wash the cells and centrifuge at 400×CF for 5 minutes. Aspirate the supernatant and resuspend the cells in 5 mL of warm CM2 supplemented with 3000 IU/mL IL-2.

Take two aliquots (20 μL each) for cell counting using an automated cell counter. Record the counts. During counting, place a 50 mL conical tube containing TIL cells in a humidified 37°C, 5% _CO2 incubator with the cap loosened to allow gas exchange. Determine the cell concentration and dilute the TIL to 1 × ^10⁶ cells/mL in CM₂ supplemented with 3000 IU/mL IL-2.

In a humidified 37°C incubator, culture at 2 ml/well in multiple wells of a 24-well tissue culture plate as needed until day 0 of the micro-REP. Culture different TIL lines in separate 24-well tissue culture plates to avoid confusion and potential cross-contamination.

Day 0, start the micro-REP . Prepare sufficient CM2 medium for the number of feeder cell batches to be tested. (For example, prepare 800 mL of CM2 medium for testing 4 feeder cell batches at once). Divide a portion of the CM2 prepared above into equal parts and supplement it with 3000 IU/mL IL-2 for cell culture. (For example, prepare 500 mL of CM2 medium with 3000 IU/mL IL-2 for testing 4 feeder cell batches at once).

To prevent cross-contamination, the 24-well plates containing TIL cultures were removed from the incubator and transferred to the BSC.

Using sterile pipettes or 100-1000 μL pipettes and tips, remove approximately 1 mL of culture medium from each well to be used and place it into an unused well of a 24-well tissue culture plate.

Using fresh, sterile pipettes or 100-1000 μL pipettes and tips, mix the remaining culture medium with the TIL in the wells to resuspend the cells, and then transfer the cell suspension to 50 mL conical tubes labeled with the TIL batch name and record the volume.

Wash each well with the reserved culture medium and transfer the volume to the same 50 mL conical tube. Rotate the cells at 400 × CF to collect the cell pellet. Drain the culture supernatant and resuspend the cell pellet in 2–5 mL of CM2 medium containing 3000 IU/mL IL-2. The volume to be used is based on the number of wells collected and the size of the pellet; that is, the volume should be sufficient to ensure a concentration >1.3 × ^10⁶ cells/mL.

Using a serum pipette, thoroughly mix the cell suspension and record the volume. Take 200 μL for cell counting using an automated cell counter. During counting, place a 50 mL conical tube containing TIL cells in a humidified 5% _CO2 incubator at 37°C with the cap loosened to allow gas exchange. Record the count.

Remove the 50 mL conical tube containing TIL cells from the incubator and resuspend the cells at a concentration of 1.3 × ^10⁶ cells/mL in warm CM2 supplemented with 3000 IU/mL IL-2. Place the 50 mL conical tube back into the incubator and loosen the cap.

For the second TIL system, repeat the above steps.

Before plating TIL into the T25 culture flasks used for experiments, dilute TIL 1:10 to a final concentration of 1.3 × ^10⁵ cells/mL as shown below.

Prepare a working solution of pure CD3 (OKT3) from MACS GMP . Remove the OKT3 stock solution (1 mg/mL) from the 4°C freezer and place it in BSC. Use OKT3 at a final concentration of 30 ng/mL in the micro-REP medium.

In each T25 culture flask used in the experiment, 600 ng OKT3 is required per 20 mL; this is equivalent to 60 μL of 10 μg/mL solution per 20 mL, or 360 μL for all 6 culture flasks tested for each feeder cell batch.

For each feeder cell batch tested, prepare 400 μL of a 1:100 dilution of 1 mg/mL OKT3 for a working concentration of 10 μg/mL (e.g., for a single test of 4 feeder cell batches, prepare 1600 μL of a 1:100 dilution of 1 mg/mL OKT3: 16 μL of 1 mg/mL OKT3 + 1.584 mL of CM2 medium with 3000 IU/mL IL-2).

Prepare T25 culture flasks . Label each culture flask and fill it with CM2 medium before preparing feeder cells. Place the flasks in a humidified 5% _CO2 incubator at 37°C to keep the medium warm while awaiting the addition of the remaining components. After preparing feeder cells, add the components to the CM2 in each culture flask.

Other information is provided in Table 36.

Table 36. Solution Information.

Prepare feeder cells . For this protocol, each batch tested requires at least 78 × ^10⁶ feeder cells. Each 1 mL vial frozen by SDBB has 100 × ^10⁶ viable cells at freezing. Assuming a 50% recovery rate after thawing from a liquid _N₂ reservoir, it is recommended to thaw at least two 1 mL vials of feeder cells per batch, thus using an estimated 100 × ^10⁶ viable cells per REP. Alternatively, if supplied in 1.8 mL vials, a single vial may provide sufficient feeder cells.

Before thawing the feeder cells, pre-warm approximately 50 mL of IL-2-free CM2 for each feeder cell batch to be tested. Remove the designated feeder cell batch vial from the LN2 reservoir, place it in a zip-lock storage bag, and keep it on ice. Thaw the vial in the sealed zip-lock storage bag by immersion in a 37°C water bath. Remove the vial from the zip-lock bag, spray or wipe it with 70% EtOH, and transfer it to a BSC.

Using a pipette, immediately transfer the contents of the feeder cell vial to 30 mL of warm CM2 in a 50 mL conical tube. Wash the vial with a small volume of CM2 to remove any residual cells and centrifuge at 400×CF for 5 min. Aspirate the supernatant and resuspend the cells in 4 mL of warm CM2 with 3000 IU/mL IL-2. Take 200 μL for cell counting using an automated cell counter. Record the count.

Cells were resuspended at 1.3 × ^10⁷ cells/mL in warm CM2 with 3000 IU/mL IL-2. TIL cells were diluted from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL.

Set up the co-culture . Dilute TIL cells from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL. Add 4.5 mL of CM2 medium to a 15 mL conical tube. Remove the TIL cells from the incubator and resuspend them thoroughly using a 10 mL serum pipette. Take 0.5 mL of the 1.3 × ^10⁶ cells/mL TIL suspension and add it to 4.5 mL of medium in a 15 mL conical tube. Return the TIL stock solution vial to the incubator. Mix thoroughly. Repeat the above steps for the second TIL line.

Transfer the culture flasks containing pre-warmed medium for a single feeder cell batch from the incubator to the BSC. Mix the feeder cells by pipetting several times up and down with a 1 mL pipette tip, and transfer 1 mL (1.3 × ^10⁷ cells) to each culture flask containing the feeder cell batch. Add 60 μL of OKT3 working stock solution (10 μg/mL) to each culture flask. Return the two control flasks to the incubator.

Transfer 1 mL (1.3 × ^10⁵ ) of each TIL batch to a correspondingly labeled T25 culture flask. Place the flask back into the incubator and incubate upright. Begin the intervention on day 5. Repeat this procedure for all feeder cell batches tested.

On day 5, the culture medium was changed . CM2 with 3000 IU/mL IL-2 was prepared. 10 mL was needed for each culture flask. Using a 10 mL pipette, 10 mL of warm CM2 with 3000 IU/mL IL-2 was transferred to each culture flask. The culture flasks were returned to the incubator and incubated upright until day 7. The above procedure was repeated for all tested feeder cell batches.

Day 7, collect the culture . Remove the culture flasks from the incubator and transfer them to BSC, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove 10 mL of culture medium from each test flask and 15 mL of culture medium from each control flask.

Using a 10 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using the appropriate standard operating procedure with an automated cell counter. Record the counts on day 7. Repeat this procedure for all feeder cell batches tested.

The replication capacity of the feeder cell control flasks was insufficient, and the fold expansion of the culture flasks containing TILs was evaluated starting from day 0.

On day 7, continue processing the feeder cell control bottles until day 14. After counting the feeder cells on day 7, add 15 mL of fresh CM2 medium containing 3000 IU/mL IL-2 to each control bottle. Return the control bottles to the incubator and incubate them upright until day 14.

Day 14, extended non-proliferative phase of the feeder cell control flasks . Remove the culture flasks from the incubator and transfer them to BSCs, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove approximately 17 mL of culture medium from each control flask. Using a 5 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. Record the volume of each culture flask.

After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using an automated cell counter device following the appropriate standard operating procedure and record the count. Repeat this procedure for all feeder cell batches tested.

B. Results and Acceptance Criteria

Results . The dose of gamma irradiation was sufficient to prevent feeder cells from replicating. All batches were expected to meet the evaluation criteria and also showed a reduction in the total number of viable feeder cells remaining on day 7 of REP culture compared to day 0. All feeder cell batches were expected to meet the following evaluation criteria: 100-fold growth expansion of TILs up to day 7 of REP culture. The feeder cell control flask counts on day 14 were expected to continue the non-proliferative trend visible on day 7.

Acceptance Criteria . Each replicated TIL line for each batch of feeder cells must meet the following acceptance criteria. The acceptance criteria are doubled, as shown in Table 37 below.

Table 37. Implementation plan for acceptance criteria.

The study evaluated whether the irradiation dose was sufficient to prevent MNC feeder cells from replicating when cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. Insufficient replication capacity was assessed by total live cell count (TVC) as determined by automated cell counting at days 7 and 14 of the repetitive period (REP).

The acceptance criterion was "no growth," meaning that on days 7 and 14, the total number of viable cells did not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP.

The ability of feeder cells to support TIL expansion was evaluated. TIL growth was measured by the fold increase in live cells from day 0 of the Reproductive Phase (REP) to day 7 of the REP. On day 7, the TIL culture reached a minimum 100-fold expansion (i.e., more than 100 times the total number of live TIL cells introduced into the culture on day 0 of the REP), as evaluated by automated cell counting.

Emergency testing of MNC feeder cell batches that do not meet acceptance criteria . If an MNC feeder cell batch fails to meet any of the acceptance criteria outlined above, the following steps will be performed to retest the batch to rule out simple experimenter error as the cause.

If two or more satellite testing vials remain in the batch, then the batch shall be tested again. If one satellite testing vial remains in the batch, or if no satellite testing vials remain in the batch, then the batch is deemed unqualified according to the acceptance criteria listed above.

For the materials to be considered合格 (qualified), both the relevant batch and the control batch must meet the above acceptance criteria. Only after meeting these criteria is the batch permitted for use.

Example 4: Preparation of IL-2 stock solution

This embodiment describes a process for dissolving purified lyophilized recombinant human interleukin-2 in a stock sample suitable for use in other tissue culture protocols (including all those described in this application and in the embodiments), including those involving the use of rhIL-2.

Procedure. Prepare a 0.2% acetic acid solution (HAc). Transfer 29 mL of sterile water to a 50 mL conical tube. Add 1 mL of 1N acetic acid to the 50 mL conical tube. Mix thoroughly by inverting the tube 2-3 times. Sterilize the HAc solution by filtration using a Steriflip filter.

Prepare PBS containing 1% HSA. Add 4 mL of 25% HSA stock solution to 96 mL of PBS in a 150 mL sterile filter unit. Filter the solution. Store at 4°C. Complete the form for each vial of rhIL-2 prepared.

Prepare a stock solution of rhIL-2 (final concentration of 6 × ^10⁶ IU/mL). Each batch of rhIL-2 is different, and the required information can be found in the manufacturer's Certificate of Analysis (COA), such as: 1) the mass of rhIL-2 per vial (mg), 2) the specific activity of rhIL-2 (IU/mg), and 3) the recommended 0.2% HAc reduction volume (mL).

Calculate the required volume of 1% HSA for a batch of rhIL-2 using the following formula:

For example, according to the COA of rhIL-2 batch 10200121 (Cellgenix), the specific activity of a 1 mg vial is 25 × ^10⁶ IU/mg. It is recommended to reconstitute rhIL-2 in 2 mL of 0.2% HAc.

Wipe the rubber stopper of the IL-2 vial with an alcohol swab. Using a 16G needle attached to a 3mL syringe, inject the recommended volume of 0.2% HAc into the vial. Be careful not to remove the stopper while withdrawing the needle. Invert the vial three times and rotate until all powder dissolves. Carefully remove the stopper and place it on the alcohol swab. Add the calculated volume of 1% HSA to the vial.

Store rhIL-2 solution. For short-term storage (<72 hours), store vials at 4°C. For long-term storage (>72 hours), aliquot the vials into smaller volumes and store them in frozen vials at -20°C until ready for use. Avoid freeze/thaw cycles. Expiry date is 6 months after preparation date. The Rh-IL-2 label includes the supplier and catalog number, lot number, expiry date, operator initials, concentration, and aliquot volume.

Example 5: Cryopreservation Process

This embodiment describes a cryopreservation process for TILs prepared according to the procedures described herein, using a CryoMed controlled-rate cryostat (Thermo Scientific) of model 7454.

The equipment used is as follows: aluminum box holder (compatible with CS750 freezer bags), freezer box for 750mL bags, low-pressure (22psi) liquid nitrogen tank, freezer, thermocouple sensor (for the strip type of bag) and CryoStore CS750 freezer bags (OriGen Scientific).

From nucleation to -20°C, the freezing process provides a rate of 0.5°C and a cooling rate of 1°C/min until the final temperature of -80°C. The program parameters are as follows: Step 1—Wait at 4°C; Step 2: 1.0°C/min (sample temperature) to -4°C; Step 3: 20.0°C/min (chamber temperature) to -45°C; Step 4: 10.0°C/min (chamber temperature) to -10.0°C; Step 5: 0.5°C/min (chamber temperature) to -20°C; and Step 6: 1.0°C/min (sample temperature) to -80°C.

Example 6: Tumor amplification process using a defined culture medium

The procedures disclosed above can be performed using appropriate culture media (e.g., CTS ^™ OpTmizer ^™ T-cell expansion SFM, ThermoFisher, including, for example, DM1 and DM2) instead of CM1 and CM2 media.

Example 7: Exemplary GEN 2 preparation of cryopreserved TIL cell therapy

This embodiment describes cGMP manufacturing of Iovance Biotherapeutics' TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices. This embodiment describes an exemplary cGMP manufacturing of a TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices.

Table 38. Exemplary plans for process expansion.

Table 39. Culture Flask Volume

Day 0, CM1 medium preparation. In BSC, add reagents to RPMI 1640 culture bottles. Add the following reagents per bottle: heat-inactivated human AB serum (100.0 mL); GlutaMax ^™ (10.0 mL); gentamicin sulfate, 50 mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL).

Remove unnecessary materials from the BSC. Dispense culture medium reagents from the BSC, leaving gentamicin sulfate and HBSS in the BSC for use in preparing wash medium.

Thaw the IL-2 aliquot. Thaw a 1.1 mL IL-2 aliquot (6 × ^10⁶ IU/mL) (BR71424) until all ice has melted. Record the IL-2 batch number and expiration date.

Transfer the IL-2 stock solution to the culture medium. In BSC, transfer 1.0 mL of the IL-2 stock solution to the prepared CM1 Day 0 culture medium bottle. Add one bottle of CM1 Day 0 culture medium and 1.0 mL of IL-2 (6 × 10⁶ IU/mL).

Transfer G-REX100MCS to BSC. Aseptically transfer G-REX100MCS (W3013130) to BSC.

Pump all complete CM1 Day 0 culture medium into G-REX100MCS culture flasks. Alternatively, use conical tubes or G-Rex100MCS for tissue fragments.

Day 0, preparation of tumor washing medium. In BSC, 5.0 mL of gentamicin (W3009832 or W3012735) was added to a 1×500 mL HBSS medium (W3013128) bottle. Each bottle contained: HBSS (500.0 mL); gentamicin sulfate, 50 mg/mL (5.0 mL). Filtered HBSS containing gentamicin was prepared via a 1 L 0.22 μm filter unit (W1218810).

Day 0 Tumor Treatment. Obtain tumor samples and immediately transfer them to a kit at 2–8°C for treatment. Aliquot the tumor washing medium. Tumor washing 1 was performed using 8” forceps (W3009771). The tumor was removed from the sample vial and transferred to the prepared “Wash 1” culture dish. This was followed by tumor washing 2 and tumor washing 3. The tumor was measured and evaluated. It was assessed whether >30% of the total tumor area was observed to be necrotic and/or fatty tissue. Clean dissection was performed where applicable. If the tumor was large and >30% of the tissue surface was observed to be necrotic/fatty, “cleaning segmentation” was performed by removing the necrotic/fatty tissue using a combination of scalpel and/or forceps while preserving the internal structure of the tumor. The tumor was dissected. Using a combination of scalpel and/or forceps, the tumor sample was cut into an even number of appropriately sized fragments (up to 6 intermediate fragments). Intermediate tumor fragments were transferred. The tumor fragments were segmented into pieces approximately 3×3×3 mm in size. The intermediate fragments were stored to prevent dehydration. The intermediate fragment segmentation was repeated. The number of small pieces collected was determined. If only the desired tissue was preserved, additional favorable tumor fragments were selected from the “Favorable Intermediate Fragments” 6-well plate to fill the discarded fragments, so that up to 50 fragments were collected.

Prepare the conical tube. Transfer the tumor slice to a 50 mL conical tube. Prepare the BSC for G-REX100MCS. Remove the G-REX100MCS from the incubator. Aseptically transfer the G-REX100MCS culture flask to the BSC. Add the tumor fragment to the G-REX100MCS culture flask. Distribute the fragments evenly.

Incubate G-REX100MCS with the following parameters: G-REX culture flask: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Calculate: Incubation time; Lower limit = Incubation time + 252 hours; Upper limit = Incubation time + 276 hours.

After the process is complete, discard all remaining warm culture medium and thaw the aliquots of IL-2.

Day 11 - Culture medium preparation. Incubator monitoring. Incubator parameters: Temperature LED display: 37.0±2.0℃; CO2 percentage: 5.0±1.5% CO2.

Incubate 3 x 1000 mL RPMI 1640 medium (W3013112) bottles and 3 x 1000 mL AIM-V (W3009501) bottles in an incubator for ≥30 minutes. Remove the RPMI 1640 medium from the incubator. Prepare the RPMI 1640 medium. Filter the medium. Thaw 3 x 1.1 mL IL-2 aliquots (6 x 10⁶ IU/mL) (BR71424). Remove the AIM-V medium from the incubator. Add IL-2 to the AIM-V. Aseptically transfer the medium to the BSC using a 10 L Labtainer bag and relay pump transfer device.

Prepare a 10L Labtainer culture medium bag. Prepare a Baxa pump. Prepare another 10L Labtainer culture medium bag. Pump the culture medium into the 10L Labtainer. Remove the automatic pump tubing from the Labtainer bag.

Mix the culture medium. Gently knead the bag to mix. Sampling the culture medium according to the sampling plan. Take 20.0 mL of culture medium and place it in a 50 mL conical tube. Prepare the cell counting dilution tubes. In the BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" and lot number to four 15 mL conical tubes. Transfer the reagents from the BSC to 2–8°C. Prepare the 1 L transfer pack. On the outside of the BSC, fuse the 1 L transfer pack (according to process note 5.11) to the transfer device attached to the prepared "Complete CM2 Day 11 Medium" bag. Prepare the feeder cell transfer pack. Incubate the Complete CM2 Day 11 Medium.

Day 11 - TIL collection. Pretreatment form. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX100MCS from the incubator. Prepare 300mL transfer packs. Fuse the transfer packs to the G-REX100MCS.

Prepare the culture flask for TIL collection and begin the collection. Collect TILs. Using GatheRex, transfer the cell suspension through a blood filter to a 300 mL transfer pack. Examine the adherent cells on the membrane.

Rinse the culture flask membrane. Close the clamps on the G-REX100MCS. Ensure all clamps are closed. Heat-seal the TIL and "supernatant" transfer pack. Calculate the volume of the TIL suspension. Prepare the supernatant transfer pack for sampling.

Draw Bac-T samples. In the BSC, aspirate approximately 20.0 mL of supernatant from the 1 L “Supernatant” transfer pack and dispense into sterile 50 mL conical tubes.

Inoculate with BacT according to the sampling plan. Using an appropriately sized syringe, take 1.0 mL of sample from a prepared 50 mL conical tube labeled with BacT and inoculate it into an anaerobic bottle.

Incubate the TIL. Place the TIL transfer kit in an incubator only when needed. Perform cell counting and calculations. Measure the mean viable cell concentration and mean cell viability for each cell count. Viability ÷ 2. Viable cell concentration ÷ 2. Measure the upper and lower limits of the count. Lower limit: mean viable cell concentration × 0.9. Upper limit: mean viable cell concentration × 1.1. Confirm that both counts are within acceptable limits. Measure the mean viable cell concentration based on all four counts performed.

Adjusting the volume of the TIL suspension: Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume (A). Volume of the removed cell count sample (4.0 mL) (B) Adjusted total TIL cell volume C = A - B.

Calculate the total number of viable TIL cells. Average viable cell concentration*: total volume; total number of viable cells: C = A × B.

Flow cytometry calculation: If the total count of live TIL cells is ≥4.0× ^10⁷ , then calculate the volume of 1.0× ^10⁷ cells for the flow cytometry sample.

Total number of live cells required for flow cytometry: 1.0 × ^10⁷ cells. Cell volume required for flow cytometry: live cell concentration divided by 1.0 × ^10⁷ cells A.

Calculate the volume of a TIL suspension equal to 2.0 × ^10⁸ live cells. Calculate the volume of excess TIL cells to be removed as needed, and remove the excess TIL and place it in an incubator as needed. Calculate the total amount of excess TIL to be removed as needed.

The calculated volume of CS-10 medium added to the excess TIL cells at the target cell concentration for freezing is 1.0 × ^10⁸ cells/mL. Centrifuge the excess TIL as needed. Observe the conical tube and add CS-10.

Fill vials. Divide 1.0 mL of cell suspension into appropriately sized vials. Divide the remaining volume into appropriately sized vials. If the volume is ≤0.5 mL, add CS10 to the vials until the volume is 0.5 mL.

Calculate the cell volume required for 1 × ^10⁷ cells for cryopreservation. Remove the sample for cryopreservation. Place the TIL in an incubator.

Cryopreservation of samples. Observe the conical tube and slowly add CS-10, recording the volume of 0.5 mL of CS-10 added.

Day 11 - Feeder Cells. Obtain feeder cells. Obtain at least two bags of feeder cells from two different batches using an LN2 freezer. Preserve the cells on dry ice before thawing. Prepare a water bath or cryotherm. Thaw the feeder cells in a water bath or cryotherm at 37.0 ± 2.0 °C for approximately 3–5 minutes or until the ice has just melted. Remove the culture medium from the incubator. Combine the thawed feeder cells. Add the feeder cells to the transfer packs. Dispense the feeder cells from the syringe into the transfer packs. Mix the combined feeder cells and label the transfer packs.

Calculate the total volume of the feeder cell suspension in the transfer pack. Remove the cell counting samples. Using a separate 3 mL syringe, aspirate 4 × 1.0 mL of cell counting sample from the feeder cell suspension transfer pack using the non-essential injection port for each sample. Aliquot each sample into labeled cryovials. Perform cell counting, determine the multiplier, select a protocol, and enter the multiplier. Measure the mean viable cell concentration and mean cell viability for the cell count. Determine the upper and lower limits of count and confirm that they are within these limits.

Adjust the volume of the feeder cell suspension. Calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Calculate the total number of viable feeder cells. Obtain additional feeder cells as needed. Thaw additional feeder cells as needed. Place the fourth feeder cell bag in a zip-lock bag and thaw in a 37.0±2.0°C water bath or Cytotherm for approximately 3–5 minutes, and combine with the additional feeder cells. Measure the volume. Measure the volume of feeder cells in the syringe and record it below (B). Calculate the new total volume of feeder cells. Add the feeder cells to the transfer pack.

Prepare diluents as needed, adding 4.5 mL of AIM-V medium to four 15 mL conical tubes. Prepare for cell counting. Using a separate 3 mL syringe for each sample, remove 4 × 1.0 mL of the cell count sample from the feeder cell suspension transfer pack using the non-essential injection port. Perform cell counting and calculations. Determine the mean viable cell concentration based on all four counts performed. Adjust the feeder cell suspension volume and calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Subtract the removed 4.0 mL from the total feeder cell volume. Calculate the volume of feeder cell suspension required to obtain 5 x ^10⁹ viable feeder cells. Calculate the excess feeder cell volume. Calculate the volume of excess feeder cells to be removed. Remove the excess feeder cells.

Using a 1.0 mL syringe and a 16G needle, aspirate 0.15 mL of OKT3 and add OKT3. Heat-seal the feeder cell suspension transfer pack.

Day 11: G-REX Filling and Inoculation Setup for G-REX500MCS. Remove the "Complete CM2 Day 11 Medium" from the incubator and pump the medium into the G-REX500MCS. Pump 4.5L of medium into the G-REX500MCS, filling to the marked line on the culture flask. Heat seal as needed and incubate the culture flask. Fuse the feeder cell suspension transfer kit to the G-REX500MCS. Add feeder cells to the G-REX500MCS. Heat seal. Fuse the TIL suspension transfer kit to the culture flask. Add TIL to the G-REX500MCS. Heat seal. Incubate the G-REX500MCS at 37.0±2.0℃, CO2 percentage: 5.0±1.5% CO2.

Calculate the incubation window. Perform calculations to determine the appropriate time to remove the G-REX500MCS from the incubator on day 16. Lower limit: Incubation time + 108 hours. Upper limit: Incubation time + 132 hours.

Day 11: Cryopreservation of Excess TIL. Applicable to: Cryopreservation of excess TIL vials. Confirm that the Controlled Rate Freezer (CRF) has been set before freezing. Perform cryopreservation. Transfer the vial from the controlled rate freezer to the appropriate storage container. After freezing is complete, transfer the vial from the CRF to the appropriate storage container. Transfer the vial to the appropriate storage container. Record the storage location in LN2.

Day 16 Culture Medium Preparation. Preheat AIM-V medium. Calculate the warming time for culture bags 1, 2, and 3. Ensure all bags have been warmed for 12 to 24 hours. Set up the 10L Labtainer for supernatant. Attach the larger diameter end of the fluid pump transfer device to a concave port of the 10L Labtainer bag using a Luer connector. Set up and label the 10L Labtainer for supernatant. Set up the 10L Labtainer for supernatant. Ensure all clamps are closed before removing from the BSC. Note: Use the supernatant bag during TIL collection, which can be performed in parallel with culture medium preparation.

Thaw IL-2. Thaw 5 × 1.1 mL IL-2 aliquots (6 × ^10⁶ IU/mL) (BR71424) of each CTS AIM V medium bag until all ice melts. Aliquot 100.0 mL GlutaMax ^™ . Add IL-2 to GlutaMax ^™ . Prepare the CTS AIM V medium bag for preparation. Prepare the CTS AIM V medium bag for preparation. Multistage Baxa pump. Prepare the medium. Pump GlutaMax ^™ + IL-2 into the bag. Monitor parameters: Temperature LED display: 37.0 ± 2.0 °C, _CO₂ percentage: 5.0 ± 1.5% _CO₂ . Warm up the complete CM4 day 16 medium. Prepare the dilution.

Day 16, REP splitting. Incubator parameters monitored: Temperature LED display: 37.0±2.0℃, _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX500MCS from the incubator. Prepare a 1L transfer pack, label it TIL suspension, and weigh it as 1L.

The volume of the G-REX500MCS decreased. Approximately 4.5 L of culture supernatant was transferred from the G-REX500MCS to a 10 L Labtainer.

Prepare the culture flask for TIL collection. After removing the supernatant, close all clamps leading to the red tubing.

Initiate TIL collection . Vigorously tap the culture flask and rotate the culture medium to detach the cells, ensuring all cells are detached.

TIL Collection . Loosen all clamps leading to the TIL suspension transfer pack. Using GatheREX, transfer the cell suspension into the TIL suspension transfer pack. Note: Ensure the edges are kept tilted until all cells and culture medium are collected. Check for adherent cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS. Heat-seal the transfer pack containing TIL. Heat-seal the 10L Labtainer containing the supernatant. Record the weight of the transfer pack containing the cell suspension and calculate the suspension volume. Prepare the transfer pack for sample removal. Remove the test sample from the cell supernatant.

Aseptic and BacT test sampling . Take 1.0 mL of sample from the prepared 15 mL cone-labeled BacT. Take the cell counting sample. In the BSC, use a separate 3 mL syringe to take 4 × 1.0 mL cell counting samples from the TIL suspension transfer pack for each sample.

Remove the Mycoplasma sample. Using a 3 mL syringe, remove 1.0 mL from the TIL suspension transfer pack and place it into a prepared 15 mL conical tube labeled “Mycoplasma diluent”.

Prepare the transfer kit for inoculation. Place the TIL in an incubator. Remove the cell suspension from the BSC and place it in an incubator until needed. Perform cell counting and calculation. First, dilute the cell count sample to a 1:10 dilution by adding 0.5 mL of the cell suspension to 4.5 mL of prepared AIM-V medium. Measure the mean viable cell concentration and mean survival rate of the cells used for cell counting. Measure the upper and lower limits of counting. Note: Dilutions can be adjusted based on the expected cell concentration. Determine the mean viable cell concentration from all four counts performed. Adjust the volume of the TIL suspension. Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume minus the 5.0 mL removed for testing.

Calculate the total number of viable TIL cells. Calculate the total number of culture flasks to be inoculated. Note: The maximum number of G-REX500MCS culture flasks to be inoculated is five. If the calculated number of culture flasks to be inoculated exceeds five, inoculate only five culture flasks using all available volumes of cell suspension.

Calculate the number of culture flasks needed for subculture. Calculate the number of culture medium bags still needed besides the prepared bags. Prepare one 10L "CM4 Day 16 Medium" bag for every two G-REX-500M culture flasks as calculated. Continue inoculating the first G-REX-500M culture flask while preparing additional medium and warming it. Prepare the determined number of additional culture medium bags and warm them. Fill the G-REX500MCS. Prepare the pumpable medium and pump 4.5L of medium into the G-REX500MCS. Heat seal. Repeat filling. Incubate the culture flasks. Calculate the target volume of TIL suspension to be added to the new G-REX500MCS culture flasks. If the calculated number of culture flasks exceeds five, inoculate only five culture flasks using all volumes of cell suspension. Prepare the culture flasks for inoculation. Remove the G-REX500MCS from the incubator. Prepare the G-REX500MCS for pumping. Close all clamps except for the larger filter line. Remove the TIL from the incubator. Prepare the cell suspension for inoculation. Aseptically fuse the "TIL suspension" transfer package (according to process note 5.11) to the pump inlet line. Place the TIL suspension bag on the scale.

Inoculate the culture flasks with TIL suspension. Pump the calculated volume of TIL suspension into the culture flasks. Heat seal. Fill the remaining culture flasks.

Monitoring the incubator. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Incubation culture bottles.

The time range for removing G-REX500MCS from the incubator on day 22 was determined.

Day 22 Wash Buffer Preparation. Prepare a 10L Labtainer bag. Attach a 4” plasma transfer device to the 10L Labtainer bag via a Luer connector in the BSC. Prepare the 10L Labtainer bag. Close all clamps before transferring out of the BSC. Note: Prepare one 10L Labtainer bag for every two G-REX 500MCS culture flasks to be collected. Aspirate the Plasmalyte pump into the 3000mL bag and remove air from the 3000mL Origen bag by inverting the pump and manipulating the bag's position. Add 25% human albumin to the 3000mL bag. Obtain a final volume of 120.0mL of 25% human albumin.

Prepare the IL-2 diluent. Using a 10 mL syringe, remove 5.0 mL of LOVO wash buffer through the needle-free injection port on the LOVO wash buffer bag. Dispense the LOVO wash buffer into 50 mL conical tubes.

Distribute LOVO wash buffer into equal portions of the CRF blank bags. Using a 100mL syringe, draw 70.0mL of LOVO wash buffer from the needleless injection port.

Thaw one 1.1 mL IL-2 (6 × 10⁶ IU/mL) until all ice has melted. Add 50 μL of IL-2 stock solution (6 × ^10⁶ IU/mL) to a 50 mL conical tube labeled “IL-2 Diluent”.

Preparation for cryopreservation . Place 5 cryopreservation boxes at 2-8°C to pretreat them for use in the final product cryopreservation.

Prepare the cell counting dilution. In a BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" to four separate 15 mL conical tubes. Prepare for cell counting. Label the four frozen vials with vial numbers (1-4). Store the vials in the BSC for later use.

TIL collection on day 22. Monitor the incubator. Incubator parameters: Temperature LED display: 37±2.0℃, CO2 percentage: 5%±1.5%. Remove the G-REX500MCS culture flask from the incubator. Prepare and label the TIL collection bag. Seal any additional connectors. Volume reduction: Transfer approximately 4.5L of supernatant from the G-REX500MCS to the supernatant bag.

Prepare the culture flask for TIL collection. Initiate TIL collection. Vigorously tap the culture flask and rotate the culture medium to detach the cells. Ensure all cells have detached. Initiate TIL collection. Loosen all clamps leading to the TIL suspension collection bag. TIL collection. Using GatheRex, transfer the TIL suspension to a 3000 mL collection bag. Examine the adhered cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS, ensuring all clamps are closed. Transfer the cell suspension to the LOVO source bag. Close all clamps. Heat seal. Remove 4 × 1.0 mL cell counting samples.

Perform cell counting. Cell counting and calculation were performed using NC-200 and process note 5.14. The cell counting sample was first diluted by adding 0.5 mL of cell suspension to 4.5 mL of prepared AIM-V medium, resulting in a 1:10 dilution. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The upper and lower limits of counting were determined. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The LOVO source bag was weighed. The total number of viable TIL cells was calculated. The total number of nucleated cells was calculated.

Prepare the Mycoplasma diluent. Take 10.0 mL from a supernatant bag via the Luhr sample port and place it into a 15 mL conical tube.

Perform the "TIL G-REX collection" protocol and determine the target volume of the final product. Load the disposable kit. Remove the filtrate bag. Enter the filtrate volume. Place the filtrate container on the lab bench. Attach PlasmaLyte. Confirm that PlasmaLyte has been attached and that it is moving. Attach the source container to the tubing and confirm that it has been attached. Confirm that PlasmaLyte is moving.

Final preparation and filling. Target volume/bag calculation. Calculate the volume of CS-10 and LOVO wash buffer to be prepared in blank bags. Prepare CRF blank bags.

Calculate the volume of IL-2 to be added to the final product. Required final IL-2 concentration (IU/mL) - 300 IU/mL. IL-2 working stock solution: 6 × ^10⁴ IU/mL. Assemble and connect the device. Aseptically fuse the 4S-4M60 to the CC2 unit connector. Aseptically fuse the CS750 cryoprotectant bag to the prepared wire bundle. Fuse the CS-10 bag to the tip of the 4S-4M60. Prepare TILs with IL-2. Using an appropriately sized syringe, extract the measured amount of IL-2 from the “IL-2 6 × ^10⁴ ” aliquot. Label the prepared TIL bag. Add the prepared TIL bag to the device. Add CS10. Switch syringes. Aspirate approximately 10 mL of air into a 100 mL syringe and replace the 60 mL syringe on the device. Add CS10. Prepare the CS-750 bag. Dispense cells.

Remove air from the final product bag and obtain the retention. Once the last final product bag has been filled, close all clamps. Draw 10 mL of air into a new 100 mL syringe and replace the syringe on the device. Dispense the retention into 50 mL conical tubes and label the tubes “Retention” and the batch number. Repeat the air removal steps for each bag.

Prepare the final product for cryopreservation, including visual inspection. Store the cryopreservation bag on a cooling pack or at 2-8°C before cryopreservation.

Remove the cell counting sample. Using an appropriately sized pipette, remove 2.0 mL of the retained sample and place it in a 15 mL conical tube for cell counting. Perform cell counting and calculation. Note: Only dilute one sample to an appropriate confirmatory dilution. Dilute the remaining sample to the appropriate dilution factor and continue counting. Measure the mean viable cell concentration and mean viability of the cells used for cell counting. Measure the upper and lower limits of count. Note: The dilution may be adjusted according to the expected cell concentration. Measure the mean viable cell concentration and mean viability. Measure the upper and lower limits of count. Calculate IFN-γ. Heat-seal the final product bag.

The following exemplary sampling plan is used to label and collect samples.

Table 40. Sample Plan.

Sterility and BacT test . Test sampling. In the BSC, take 1.0 mL of sample from the retained cell suspension collected using an appropriately sized syringe and inoculate it into an anaerobic flask. Repeat the above steps for the aerobic flask.

Cryopreservation of the final product. Prepare the controlled-rate freezer (CRF). Verify that the CRF is set up. Set the CRF probe. Place the final product and sample in the CRF. Measure the time required to reach 4℃ ± 1.5℃ and continue the CRF operation. Complete the CRF and store. Stop the CRF after completion. Remove the box and vial from the CRF. Transfer the box and vial to gas chromatography-tandem nitrogen (LN2) for storage. Record the storage location.

The final processing and analysis of the drug included the following tests: (Day 22) CD3+ cell count of REP on Day 22 by flow cytometry; (Day 22) Gram staining method (GMP); (Day 22) bacterial endotoxin test by gel clot LAL assay (GMP); (Day 16) BacT sterility assay (GMP); (Day 16) TD-PCR (GMP) detection of Mycoplasma DNA; acceptable appearance characteristics; (Day 22) BacT sterility assay (GMP); (Day 22) IFN-γ assay. Other potency assays as described herein were also used to analyze the TIL product.

Example 8: Exemplary Implementation of the GEN 3 Amplification Platform

Day 0

Prepare tumor washing medium. Warm the medium before starting. Add 5 mL of gentamicin (50 mg/mL) to a 500 mL HBSS bottle. Add 5 mL of tumor washing medium to a 15 mL Erlenmeyer flask for OKT3 dilution. Prepare feeder cell bags. Aseptically transfer feeder cells to feeder cell bags and store at 37°C until use or freeze. If stored at 37°C, count the feeder cells. If frozen, thaw and then count the feeder cells.

The optimal range for feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with a 4.5 mL LAIM-V. Add 0.5 mL of cell fraction for each cell count. If the total number of viable feeder cells is ≥ 1 × ^10⁹ cells, adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag to add 1 × ^10⁹ cells to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of tumor washing medium to an OKT3 aliquot (100 μL). Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the second feeder cell bag. Adjust the medium volume to a total volume of 2 L. Transfer the second feeder cell bag to an incubator.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 0: 15 μg/vial, i.e., 30 ng/mL in 500 mL, up to approximately 60 μL, 1 aliquot.

Add 5 mL of tumor wash medium to all wells of a 6-well plate labeled as excess tumor flakes. Reserve the tumor wash medium for further use in maintaining tumor hydration during dissection. Add 50 mL of tumor wash medium to each 100 mm Petri dish.

Using a ruler as a reference, divide the tumor into ^27mm segments (3×3×3mm) under the dissecting culture dish lid. Dissect the intermediate segments until 60 segments are obtained. Count the total number of final segments and prepare G-REX-100MCS culture flasks (usually 60 segments/flask) based on the number of final segments produced.

Preserve favorable tissue fragments in conical tubes labeled Fragment Tubes 1 through 4. Calculate the number of G-REX-100MCS culture flasks seeded with feeder cell suspension based on the number of initial fragment tubes.

Remove the feeder cell bag from the incubator and inoculate with G-REX-100 MCS. Label it D0 (day 0).

Add the tumor fragment to the culture in the G-REX-100 MCS . Under aseptic conditions, unscrew the caps of the G-REX-100 MCS labeled "Tumor Fragment Culture (D0) 1" and the 50 mL conical tube labeled "Fragment Tube". Rotate the opened fragment tube 1 while simultaneously slightly lifting the cap of the G-REX-100 MCS. Add the culture medium and fragment together to the G-REX-100 MCS while rotating. Record the number of fragments transferred to the G-REX-100 MCS.

Once the fragment is at the bottom of the GREX culture flask, aspirate 7 mL of culture medium and create seven 1 mL aliquots: 5 mL for extended characterization and 2 mL for sterility. Store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until needed.

Inoculate one anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle with 1 mL of the final fragment culture supernatant. Repeat the above procedure for each culture bottle from which samples are taken.

On the 7th-8th day

Prepare the feeder cell bags. If frozen, thaw the feeder cell bags in a 37°C water bath for 3–5 minutes. If frozen, count the feeder cells. The optimal feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with 4.5 mL AIM-V. For each cell count, add 0.5 mL of the cell fraction to a new frozen tube. Mix the sample thoroughly and perform cell counting.

If the total number of live feeder cells is ≥2× ^10⁹ cells, proceed to the next step to adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag so that 2× ^10⁹ cells can be added to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of HBSS to 100 μL of OKT3 aliquot. Mix by pipetting three times, upwards and downwards. Prepare two aliquots.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 7/8: 30 μg/vial, i.e., 60 ng/mL in 500 mL, up to 120 μL, 2 aliquots.

Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the feeder cell bag, ensuring all is added. Adjust the culture medium volume to a total of 2 L. Repeat the above steps for the second OKT3 aliquot and add it to the feeder cell bag. Transfer the second feeder cell bag to an incubator.

Prepare G-REX100MCS culture flasks containing feeder cell suspension . Record the number of G-REX100MCS culture flasks to be processed based on the number of G-REX culture flasks produced on day 0. Remove the G-REX culture flasks from the incubator and remove the second feeder cell bag from the incubator.

Remove the supernatant before adding the feeder cell suspension . Connect a 10 mL syringe to a G-REX100 culture flask and aspirate 5 mL of culture medium. Prepare five 1 mL aliquots, 5 mL for extended characterization, and store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until requested by the initiator. Label each G-REX100 culture flask and repeat the above steps.

Depending on the number of culture flasks, 5-20 × 1 mL samples are used for characterization:

●5mL = 1 culture flask

●10mL = 2 culture flasks

●15mL = 3 culture flasks

●20mL = 4 culture flasks

Continue seeding feeder cells into G-REX100 MCS flasks, and repeat the above procedure for each G-REX100 MCS flask. Using aseptic transfer, transfer 500 mL of the second feeder cell bag by weight (assuming 1 g = 1 mL) to each G-REX-100 MCS flask and record the transfer volume. Label this as Day 7 culture and repeat the above procedure for each G-REX100 flask. Transfer the G-REX-100 MCS flasks to an incubator.

Days 10-11

Remove the first G-REX-100MCS culture flask and, under aseptic conditions, use a 10mL syringe to extract 7mL of pretreated culture supernatant. Prepare seven 1mL aliquots: 5mL for extended characterization and 2mL for sterile samples.

Carefully mix the culture flasks and use a new 10mL syringe to remove 10mL of the supernatant and transfer it to a 15mL tube labeled D10/11 Mycoplasma supernatant.

Carefully mix the culture flasks and, using a new syringe, dispense the following volumes based on the number of culture flasks to be processed:

● 1 bottle = 40mL

●2 bottles = 20mL/bottle

● 3 bottles = 13.3 mL/bottle

● 4 bottles = 10mL/bottle

Aspirate a total of 40 mL from all culture flasks and collect the samples in a 50 mL conical tube labeled ‘Day 10/11 QC Sample’. Store the tube in an incubator until needed. Perform cell counting and cell distribution.

Five aliquots (pretreated culture supernatant) used for extended characterization were stored at ≤-20°C until needed. One anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle were inoculated with 1 mL of pretreated culture supernatant, respectively.

Continue transferring the cell suspension to G-REX-500MCS and repeat the above procedure for each G-REX-100MCS. Under aseptic conditions, transfer the contents of each G-REX-100MCS to a G-REX-500MCS, monitoring approximately 100 mL of fluid transfer each time. Stop transferring when the volume of the G-REX-100MCS decreases to 500 mL.

During the transfer step, use a 10 mL syringe and aspirate 10 mL of cell suspension from the G-REX-100MCS into the syringe. Follow the instructions according to the number of flasks in culture. If only 1 flask: use two syringes to aspirate a total of 20 mL. If 2 flasks: aspirate 10 mL from each flask. If 3 flasks: aspirate 7 mL from each flask. If 4 flasks: aspirate 5 mL from each flask. Transfer the cell suspension to a common 50 mL conical tube. Keep in the incubator until the cell counting step and QC sample. The total number of cells required for QC is approximately 20e6 cells: 4 × 0.5 mL cell counts (cell counts are undiluted first).

The required cell quantity for the assay is as follows:

1. At least 10 × ^10⁶ cells should be used for efficacy assays, such as those described herein, or for IFN-γ or granzyme B assays.

2.1 × ^10⁶ cells were used for Mycoplasma.

3.5 × ^10⁶ cells were used for CD3+/CD45+ flow cytometry.

Transfer the G-REX-500MCS culture flask to an incubator.

Prepare QC samples . In this embodiment, at least 15 × ^10⁸ cells are required for the assay. Assays include: cell count and viability; Mycoplasma (1 × ^10⁶ cells/mean viable concentration); flow cytometry (5 × ^10⁶ cells/mean viable concentration); and IFN-γ assay (5 × ^10⁶ cells - 1 × ^10⁶ cells; IFN-γ assay requires 8-10 × ^10⁶ cells).

Calculate the volume of the cell fraction cryopreserved at 10 × ^10⁶ cells/mL, and calculate the number of vials required.

Days 16-17

Wash buffer preparation (1% HSA Plasmalyte A). Transfer the HSA and Plasmalyte to a 5L bag to prepare the LOVO wash buffer. Under aseptic conditions, transfer a total volume of 125 mL of 25% HSA to a 5L bag. Remove 10 mL or 40 mL of the wash buffer and transfer it to an 'IL-2 6× ^10⁴ IU/mL tube' (10 mL if the IL-2 is pre-prepared, or 40 mL if the IL-2 is freshly prepared).

Calculate the volume of restored IL-2 added to Plasmalyte + 1% HSA: Volume of restored IL-2 = (Final concentration of IL-2 × Final volume) / Specific activity of IL-2 (based on standard determination). The final concentration of IL-2 is 6 × ^10⁴ IU/mL. The final volume is 40 mL.

Take the calculated initial volume of IL-2 required for IL-2 recovery and transfer it to a tube labeled 'IL-2 6 × ^10⁴ IU/mL'. Add 100 μL of IL-2 6 × ^10⁶ IU/mL from the pre-prepared aliquot to a tube labeled 'IL-2 6 × ^10⁴ IU/mL' containing 10 mL of LOVO wash buffer.

Remove approximately 4500 mL of supernatant from the G-REX-500MCS culture flask. Rotate the remaining supernatant and transfer the cells to the cell collection pocket. Repeat the above steps for all G-REX-500MCS culture flasks.

Take 60 mL of the supernatant and add it to the supernatant tube for quality control determination, including mycoplasma detection. Store at +2–8°C.

Cell collection . Cell counting. Prepare four 15 mL conical tubes containing 4.5 mL of AIM-V. These conical tubes can be prepared in advance. Optimal range = between 5 × ^10⁴ and 5 × ^10⁶ cells/mL (1:10 dilution recommended). For a 1:10 dilution, add 500 μL of CF to the previously prepared 4500 μL of AIM-V. Record the dilution factor.

Calculate the total cells (TC) of pre-LOVO (survival + death) =

Average total cells

Concentration (pre-LOVO TC concentration)

(Survival + Death)

X

Volume of source bag

Calculate the TVC (total viable cells) of pre-LOVO (survival) =

Average total viable cells

Concentration (pre-LOVO TVC)

(Survival)

X

LOVO source bag volume

When the total cell count (TC) is >5 × ^10⁹ , remove 5 × ^10⁸ cells for cryopreservation as a reserve sample for MDA. 5 × ^10⁸ ÷ average TC concentration (step 14.44) = volume to be removed.

When the total cell count (TC) is ≤5× ^10⁹ , 4× ^10⁶ cells are removed and cryopreserved as a reserve sample for MDA. 4× ^10⁶ ÷ average TC concentration = volume to be removed.

When determining the total cell count, the number of cells to be removed should allow for the retention of 150 × ^10⁹ viable cells. Confirm the pre-LOVO TVC as 5 × ^10⁸ or 4 × ^10⁶ , or not applicable. Calculate the volume of cells to be removed.

Calculate the remaining total cells in the bag. Calculate the pre-LOVO TC (total cells). [Average total cell concentration x remaining volume = pre-LOVO remaining TC]

Select the corresponding process from Table 41 based on the total number of remaining cells.

Table 41. Total number of cells.

Select the volume of IL-2 to be added corresponding to the procedure used. The volume is calculated as follows: Residue volume × 2 × 300 IU/mL = Required IU of IL-2. Required IU of IL-2 / 6 × ^10⁴ IU/mL = Volume of IL-2 added after LOVO bagging. Record all added volumes. Obtain samples in frozen vials for further analysis.

Mix the cell products thoroughly. Seal all bags for further processing, including freezing where appropriate.

Perform endotoxin, IFN-γ, sterility, and other assays on the obtained frozen vial samples as needed.

Example 9: Exemplary Processes for GEN 2 and GEN 3

This example illustrates the Gen 2 and Gen 3 processes. The Gen 2 and Gen 3 TILs typically consist of autologous TILs derived from individual patients (via surgical tumor resection) and then expanded ex vivo. The first expansion step in initiating the Gen 3 process involves cell culture in the presence of interleukin-2 (IL-2) and the monoclonal antibody OKT3, which targets the T-cell co-receptor CD3 on the cytoskeleton of irradiated peripheral blood mononuclear cells (PBMCs).

The fabrication of the Gen 2 TIL product consists of two stages: 1) pre-rapid expansion (pre-REP) and 2) rapid expansion protocol (REP). During pre-REP, excised tumors are cut into ≤50 fragments of 2-3 mm in each dimension. These fragments are cultured for 11 days in serum-containing medium (RPMI 1640 medium supplemented with 10% HuSAB) and 6,000 IU/mL interleukin-2 (IL-2). On day 11, TILs are collected and introduced into a large-scale secondary REP expansion. The REP consists of the following: activation of ≤200 × ^10⁶ live cells from pre-REP for 5 days in ⁵ L of CM2 supplemented with 3,000 IU/mL rhIL-2, with the feeder cells loaded with 150 μg of monoclonal anti-CD3 antibody (OKT3). On day 16, the culture volume was reduced by 90%, and the cells were fractionated to ≥1× ^10⁹ viable lymphocytes/flask into multiple G-REX-500 culture flasks, and brought to a final volume of 5L with CM4. The TIL was then incubated for another 6 days. On day 22, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

The fabrication of Gen 3 TIL products consists of three stages: 1) initiation of the first expansion protocol, 2) rapid second expansion protocol (also known as the rapid expansion phase or REP), and 3) dissociation of subcultures. To achieve initiation of the first expansion TIL proliferation, the excised tumor was fragmented into ≤120 fragments, each 2-3 mm in dimensions. On day 0 of initiation of the first expansion, a feeder layer of approximately 2.5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was established on a surface area of approximately 100 ^cm² in each of three 100 MCS dishes. The tumor fragments were distributed in the three 100 MCS dishes and cultured for 7 days in CM1 medium containing 500 mL of serum and 6,000 IU/mL of interleukin-2 (IL-2) and 15 μg of OKT-3. On day 7, REP was initiated as follows: an additional feeder cell layer of approximately 5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was incorporated into the tumor fragmentation culture phase in each of three 100 MCS dishes and cultured with 500 mL CM2 medium and 6,000 IU/mL IL-2 and 30 μg OKT-3. REP initiation was enhanced by activating the entire initiation first expansion culture in the same dish, which was achieved by transferring OKT3-loaded feeder cells to the 100 MCS dishes using a closed-system fluid transfer. For Gen 3, TIL scaling up or splitting involved the following procedural steps: the entire cell culture was scaled up and transferred to the larger dish via a closed-system fluid transfer (from a 100 M culture flask to a 500 M culture flask) and an additional 4 L CM4 medium was added. On day 16, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

Overall, the Gen 3 process is a shorter, more scalable, and easier-to-modify amplification platform that will be suitable for stable manufacturing and process comparability.

Table 50. Comparison of the manufacturing processes of Exemplary Gen 2 and Exemplary Gen 3.

On day 0, for both processes, tumor cells were washed three times and fragments were randomly grouped into two pools; one pool for each process. For the Gen 2 process, fragments were transferred to a GREX 100MCS flask containing 1 LCM1 of medium with 6,000 IU/mL rhIL-2. For the Gen 3 process, fragments were transferred to a G-REX100MCS culture flask containing 500 mL CM1 of medium with 6,000 IU/mL rhIL-2, 15 μg OKT-3, and 2.5 × ^10⁸ feeder cells. TILs were inoculated on Rep start day according to different days for each process. For the Gen 2 process, where the volume of the G-REX-100MCS culture flasks decreased by 90%, the collected cell suspension was transferred to new G-REX-500MCS on day 11 to initiate REP in CM2 medium containing IL-2 (3000 IU/mL) plus 5 × ^10⁹ feeder cells and OKT-3 (30 ng/mL). Cells were expanded according to the protocol and split into multiple G-REX-500MCS culture flasks containing CM4 medium and IL-2 (3000 IU/mL) on day 16. Cultures were then collected and cryopreserved on day 22 according to the protocol. For the Gen 3 process, REP initiation was performed on day 7 using the same G-REX-100MCS. In short, 500 mL of CM2 medium containing IL-2 (6000 IU/mL), 5 × ^10⁸ feeder cells, and 30 μg OKT-3 was added to each culture flask. On days 9–11, scale up the culture. Transfer the entire volume of G-REX100M (1 L) to G-REX-500MCS and add 4 L of CM4 containing IL-2 (3000 IU/mL). Incubate the flask for 5 days. On day 16, collect the culture and freeze it.

The comparison included three different tumors: two lung tumors (L4054 and L4055) and one melanoma tumor (M1085T).

For L4054 and L4055, CM1 (medium 1), CM2 (medium 2), and CM4 (medium 4) media were prepared in advance and maintained at 4°C. CM1 and CM2 media were prepared without filtration to compare cell growth with and without filtration of the media.

For L4055 tumors, the culture medium was heated at 37°C for up to 24 hours during REP initiation and scaling up.

Results . For the total number of viable cells achieved, the results for Gen 3 were within 30% of those for Gen 2. Upon restimulation, the Gen 3 final product exhibited higher IFN-γ production. As measured by the total number of unique CDR3 sequences present, the Gen 3 final product showed increased clonal diversity. The Gen 3 final product also exhibited a longer mean telomere length.

Pre-REP and REP expansion in Gen 2 and Gen 3 processes followed the procedures described above. For each tumor, both pools contained an equal number of fragments. Due to the smaller size of the tumors, the maximum number of fragments per flask could not be reached. Total pre-REP cells (TVCs) were collected and counted on day 11 of the Gen 2 process and day 7 of the Gen 3 process. To compare the two pre-REP groups, the cell count was divided by the number of fragments provided in the culture to calculate the average number of viable cells per fragment. As indicated in Table 51 below, more cells were consistently grown per fragment in the Gen 2 process compared to the Gen 3 process. The extrapolation of the expected TVC number on day 11 of the Gen 3 process was calculated by dividing the pre-REP TVC by 7 and then multiplying by 11.

Table 51. Pre-REP cell count

*L4055, unfiltered culture medium.

For both Gen 2 and Gen 3 processes, TVCs were counted according to process conditions, and the percentage of viable cells per day of the process was generated. At harvest, cells were collected on day 22 (Gen 2) and day 16 (Gen 3), and TVC counts were generated. The TVCs were then divided by the number of fragments provided on day 0 to calculate the average number of viable cells per fragment. The fold increase was calculated by dividing the collected TVCs by the REP initiation TVCs. As shown in Table 52, comparing Gen 2 and Gen 3, the fold increases were similar for L4054; in the case of L4055, the fold increase was higher for the Gen 2 process. Specifically, in this case, the medium was heated to 24°C prior to the REP initiation day. A higher fold increase was also observed in Gen 3 for M1085T. The extrapolation of the expected TVC count on day 22 of the Gen 3 process was calculated by dividing the REP TVCs by 16 and then multiplying by 22.

Table 52. Total viable cell count and fold expansion of TIL final products.

*L4055, unfiltered culture medium.

Table 53: Survival percentage of final TIL products: Final TIL REP products were compared to the survival percentage release criteria after collection. All conditions for Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Following collection, the final TIL REP product was compared against release criteria based on survival percentage. All conditions for both Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Table 53. Survival Percentage of REP (TIL Final Product)

Because the number of fragments in each vial is less than the maximum required number, the estimated cell count on the collection day is calculated for each tumor. This assessment is based on the expectation that the clinical tumor is large enough on day 0 to be inoculated with 2 or 3 vials.

Table 54. Extrapolation of estimated cell counts to full-size 2 and 3 bottles for the Gen 3 process.

Immunophenotypic Analysis – Comparison of Phenotypic Markers in TIL End Products. Three tumor types, L4054, L4055, and M1085T, underwent TIL amplification during both Gen 2 and Gen 3 processes. Following collection, REP TIL end products were analyzed by flow cytometry to test for purity, differentiation, and memory markers. For all conditions, the percentage of TCR a/b+ cells exceeded 90%.

TILs collected from the Gen 3 process showed higher expression of CD8 and CD28 compared to those collected from the Gen 2 process. The Gen 2 process showed a higher percentage of CD4+.

TILs collected from the Gen 3 process showed higher expression of the central memory chamber compared to TILs collected from the Gen 2 process.

Activation and depletion biomarkers were analyzed in TILs from two tumors, L4054 and L4055, to compare the final TIL products from the Gen 2 and Gen 3 TIL amplification processes. Activation and depletion biomarkers for the Gen 2 and Gen 3 processes were similar.

Interferon-γ secretion upon restimulation. On the collection days, day 22 of Gen 2 and day 16 of Gen 3, TILs of L4054 and L4055 were restimulated overnight using coated anti-CD3 plates. M1085T was restimulated using anti-CD3, CD28, and CD137 beads. Under all conditions, the supernatant was collected and frozen 24 hours after restimulation. IFNγ analysis by ELISA was performed simultaneously on the supernatants from both processes using the same ELISA plate. Higher IFNγ production from the Gen 3 process was observed in all three tumors analyzed.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption during Gen 2 versus Gen 3 processes, cell supernatants were collected on REP initiation, scale-up, and collection days for tumors L4054 and L4055. The amount of IL-2 in the cell culture supernatant was measured using a Quantitate ELISA kit from R&D. A general trend indicates that IL-2 concentrations remained higher during Gen 3 compared to Gen 2. This may be attributed to the higher IL-2 concentration at Gen 3 REP initiation (6000 IU/mL) and residual culture medium throughout the process.

Metabolic substrate and metabolite analysis. The levels of metabolic substrates (e.g., D-glucose and L-glutamine) are measured as substitutes for overall culture medium consumption. Their reciprocal metabolites, such as lactate and ammonia, are also measured. Glucose is a monosaccharide in the culture medium, used by mitochondria to produce energy in the form of ATP. When glucose is oxidized, lactate is produced (lactate is an ester of lactate). Lactate is produced in large quantities during the exponential growth phase of cells. High levels of lactate can negatively impact cell culture processes.

For Gen 2 and Gen 3 processes, used culture media from L4054 and L4055 were collected on REP start, scale-up, and collection days. Used culture media were collected on days 11, 16, and 22 of Gen 2, and on days 7, 11, and 16 of Gen 3. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

L-Glutamine is an unstable essential amino acid required in cell culture media formulations. Glutamine contains an amine, and this amide group allows for the transport and delivery of nitrogen into cells. When L-glutamine is oxidized, cells produce toxic ammonia byproducts. To counteract L-glutamine degradation, GlutaMax ^™ was supplemented into the culture medium during the Gen 2 and Gen 3 processes, as it is more stable in aqueous solution and does not spontaneously degrade. In both tumor lines, the Gen 3 group showed a decrease in L-glutamine and GlutaMax ^™ during the process, as well as an increase in ammonia throughout the REP. In the Gen 2 group, constant L-glutamine and GlutaMax ^™ concentrations were observed, along with a slight increase in ammonia production. For ammonia, the Gen 2 and Gen 3 processes were similar at the collection day, showing only slight differences in L-glutamine degradation.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats at L4054 and L4055 was measured using Flow-FISH technology during Gen 2 and Gen 3. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Gen 3 showed similar telomere lengths to Gen 2.

CD3 analysis. To determine the clonal diversity of cell products generated in each process, the collected TIL final products from L4054 and L4055 were sampled and analyzed by sequencing the CDR3 portion of the T cell receptor for clonal diversity analysis.

Table 55 shows a comparison of the percentage of unique CDR3 sequences shared on L4054 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 199 sequences with Gen 2 final products, and 97.07% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 55. Comparison of the common uCDR3 sequence between Gen 2 and Gen 3 processes on L4054.

Table 56 shows a comparison of the percentage of unique CDR3 sequences shared on L4055 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 1833 sequences with Gen 2 final products, and 99.45% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 56. Comparison of common uCDR3 sequences between Gen 2 and Gen 3 processes on L4055.

CM1 and CM2 culture media were prepared in advance without filtration and kept at 4°C until used for tumor L4055 for Gen 2 and Gen 3 processes.

For L4055 tumors, on the REP initiation day, the culture medium was heated at 37°C for 24 hours for use in the Gen 2 and Gen 3 processes.

LDH was not measured in the supernatant collected during the process.

M1085T TIL cells were counted using a K2 cellometer.

On the tumor M1085T, samples are unavailable, such as supernatant for metabolic analysis; TIL products for activation and depletion marker analysis, telomere length and CD3-TCR vb analysis.

Conclusion . This example compares three independent donor tumor tissues based on functional quality attributes, expanded phenotypic characterization of the Gen 2 and Gen 3 processes, and culture medium consumption.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

Assuming pre-REP collection is performed on day 11 instead of day 7 and REP collection is performed on day 22 instead of day 16, extrapolated cell numbers for the Gen 3 process were calculated. In both cases, Gen 3 showed closer TVC numbers compared to the Gen 2 process, indicating that early activation enhances TIL growth.

Extrapolating values from other flasks (2 or 3) in the Gen 3 process, assuming larger tumor sizes and the maximum number of fragments required for each process as described. It was observed that collections performed on day 16 of the Gen 3 process achieved similar TVC doses compared to day 22 of the Gen 2 process. This observation is significant and indicates that early activation of the culture may reduce TIL treatment time.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

In terms of phenotypic characterization, higher CD8+ and CD28+ expression was observed in all three tumors during the Gen 3 process compared to the Gen 2 process.

Compared to the Gen 2 process, the Gen 3 process shows a slightly higher central memory compartment.

Although the Gen 3 process is shorter in duration, the Gen 2 and Gen 3 processes show similar activation and exhaustion markers.

In the three tumors analyzed, the IFNγ production on the final product of Gen 3 was 3-fold higher than that of Gen 2. This data suggests that the Gen 3 process produces a more potent and efficient TIL product compared to the Gen 2 process, which may be attributed to higher expression of CD8 and CD28 on Gen 3. Phenotypic characterization showed a positive trend in CD8+ and CD28+ expression in Gen 3 compared to the Gen 2 process in all three tumors.

The telomere lengths of the final TIL products from Gen 2 and Gen 3 are similar.

The glucose and lactate levels of the final products of Gen 2 and Gen 3 were similar, indicating that the nutrient levels on the culture medium were not affected during the Gen 3 process because no reduction removal was performed each day during the process and the overall culture medium volume was smaller compared to Gen 2.

Compared to the Gen 2 process, the processing time of the entire Gen 3 process is reduced by about half, which will significantly reduce the cost of goods sold (COG) of TIL products amplified through the Gen 3 process.

IL-2 consumption indicates the general trend of IL-2 consumption during the Gen 2 process, and that IL-2 is higher during the Gen 3 process due to the lack of removal of old culture medium.

CDR3 TCRab sequence analysis revealed high clonal diversity in the Gen 3 process.

Adding feeder cells and OKT-3 on day 0 of the pre-REP allows for early activation of TILs and enables TIL growth using the Gen 3 process.

Table 57 describes the various implementation schemes and results of the Gen 3 process compared to the current Gen 2 process.

Table 57. Exemplary Gen 3 process characteristics.

Example 10: Exemplary GEN 3 process (also known as GEN 3.1)

This embodiment describes further studies regarding the comparability between Gen 2 and Gen 3 processes for TIL amplification. The Gen 3 process is modified to include an activation step early in the process, thereby increasing the final total live cells (TVC) output while maintaining phenotypic and functional profile. As described below, the Gen 3 implementation is modified to another implementation, referred to herein as Gen 3.1.

In some implementations, the Gen 3.1 TIL manufacturing process involves four operator interventions:

1. Tumor Fragment Isolation and Activation: On day 0 of the procedure, the tumor was dissected and final fragments of approximately 3 × ³ mm were generated (up to 240 fragments in total) and cultured in 1–4 G-REX100MCS culture flasks. Each flask contained up to 60 fragments, 500 mL of CM1 or DM1 medium, supplemented with 6,000 IU rhIL-2, 15 μg OKT3, and 2.5 × 10⁸ irradiated allogeneic mononuclear cells. The cultures were incubated at 37°C for 6–8 days.

2. TIL Culture Reactivation: On days 7-8, in both cases, replenish the culture by slowly adding CM2 or DM1 medium supplemented with 6,000 IU rhIL-2, 30 μg OKT3, and 5 × ^10⁸ irradiated allogeneic monocytes. Take care not to disturb the existing cells at the bottom of the culture flask. Incubate the culture at 37°C for 3-4 days.

3. Culture scaling up: Performed on days 10-11. During culture scaling up, in both cases, transfer all contents of the G-REX100MCS to a G-REX500MCS culture flask containing 4 L of CM4 or DM2 supplemented with 3,000 IU/mL IL-2. Incubate the culture flask at 37°C for 5-6 days until harvest.

4. Collection/Washing/Preparation: On days 16-17, reduce the volume of the culture flasks and combine them. Concentrate the cells and wash them with PlasmaLyte A pH 7.4 containing 1% HSA. Prepare the washed cell suspension with CryoStor10 at a 1:1 ratio and supplement with rhIL-2 to a final concentration of 300 IU/mL.

DPs are cryopreserved and stored in gaseous liquid nitrogen via controlled-rate freezing. *Complete standard TIL media 1, 2, or 4 (CM1, CM2, CM4) can be used instead of CTS ^™ OpTmizer ^™ T cell serum-free expansion medium, also known as the defining medium (DM1 or DM2), as mentioned above.

Procedure Description. On day 0, the tumor was washed three times and then fragmented into 3×3×3 final fragments. After fragmenting the entire tumor, the final fragments were then equally randomized and divided into three pools. One randomized fragment pool was introduced into each group, with the same number of fragments added according to the three experimental matrices.

Throughout the TIL amplification process, tumor L4063 was amplified using standard culture medium, and tumor L4064 was amplified using a specific culture medium (CTS OpTmizer). The components of the culture media are described herein.

CM1 complete medium 1: RPMI + glutamine, supplemented with 2mM GlutaMax ^™ , 10% human AB serum, gentamicin (50ug/mL), and 2-mercaptoethanol (55uM). The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM2 complete medium 2: 50% CM1 medium + 50% AIM-V medium. The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM4 complete medium 4: supplemented with AIM-V containing GlutaMax ^™ (2mM). The final medium formulation was supplemented with 3000 IU/mL IL-2.

The basal ^{medium for} T cell expansion is supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L).

DM1: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 6,000 IU/mL IL-2.

DM2: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 3,000 IU/mL IL-2.

All types of culture media used, i.e., complete (CM) and defined (DM) media, were prepared in advance and kept at 4°C until the day before use, and were preheated in an incubator at 37°C for up to 24 hours prior to the treatment day.

For both tumor types, TIL cultures were reactivated on day 7. For L4063, reactivation was performed on day 10, and for L4064, scale-up was performed on day 11. Both cultures were collected and cryopreserved on day 16.

Results achieved . Cell counts and survival percentages were determined during Gen 3.0 and Gen 3.1 procedures. Amplification under all conditions followed the details described in this example.

For each tumor, the fragments were divided into three equal pools. Due to the small size of the tumors, the maximum number of fragments per bottle could not be reached. Total viable cells and cell viability were assessed under each condition for three different procedures. Cell counts were determined as TVC at reactivation on day 7, TVC at scaling up on day 10 (L4064) or day 11 (L4063), and TVC at collection on day 16/17.

Cell counts on days 7 and 10/11 were considered as FIO. Folds of expansion were calculated by dividing the TVC at day 16/17 (when collected) by the TVC at day 7 (reactivation). To compare the three groups, the TVC at the collection day was divided by the number of fragments added to the culture on day 0 to calculate the average number of viable cells per fragment.

Cell counting and viability assays were performed on L4063 and L4064. On both tumors, the Gen3.1 assay produced more cells per fragment than the Gen 3.0 assay.

Total viable cell count and fold expansion; percentage of survival during the procedure. Percentage of survival under all conditions was obtained after reactivation, scaling up, and collection. Release criteria for survival percentage were compared to final TILs after collection on day 16/17. All conditions assessed exceeded the 70% survival criterion and were similar across all procedures and tumors.

Immunophenotyping analysis – Phenotypic characterization of TIL final products. Flow cytometry analysis of the final products was performed to test purity, differentiation, and memory markers. Under all conditions, the population percentages of TCRα/β, CD4+, and CD8+ cells were constant.

Extended phenotypic analysis of REP TILs was performed. In both tumors, TIL products showed a higher percentage of CD4+ cells under Gen 3.1 conditions compared to Gen 3.0, and a higher percentage of CD28+ cells from the CD8+ population under Gen 3.0 conditions compared to Gen 3.1 conditions under both conditions.

TILs collected from Gen 3.0 and Gen 3.1 processes showed phenotypic markers similar to CD27 and CD56 expression on CD4+ and CD8+ cells, and similar CD28 expression on CD4+-gated cell populations. Comparison of memory markers on TIL final products:

Frozen samples of TIL collected on day 16 were stained for analysis. The TIL memory state was similar in the Gen 3.0 and Gen 3.1 processes. Comparison of activation and depletion markers on the final TIL products:

The activation and depletion markers of gated Gen 3.0 and Gen 3.1 processes are similar in CD4+ and CD8+ cells.

Interferon-γ secretion after restimulation. For L4063 and L4064, collected TILs were restimulated overnight using coated anti-CD3 plates. In both tumors analyzed, higher IFNγ production from the Gen 3.1 process was observed compared to the Gen 3.0 process.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption levels under all conditions and processes, cell supernatants were collected and frozen at the start of reactivation on day 7, scale-up on day 10 (L4064)/day 11 (L4063), and collection on day 16/day 17. The supernatants were then thawed and analyzed. The amount of IL-2 in the cell culture supernatant was measured according to the manufacturer's protocol.

IL-2 consumption was similar throughout the entire Gen 3 and Gen 3.1 processes, evaluated under the same culture conditions. IL-2 concentrations (pg/mL) were analyzed in the used culture media collected from L4063 and L4064.

Metabolite analysis. For each condition, used culture supernatant was collected from L4063 and L4064 at day 7 for reactivation initiation, day 10 (L4064)/day 11 (L4063) for scaling up, and day 16/day 17 for collection. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

Compared to the complete medium (2 g/L), the glucose concentration in the determined medium was higher, at 4.5 g/L. Overall, the glucose concentration and consumption were similar for the Gen 3.0 and Gen 3.1 processes across each medium type.

An increase in lactate was observed, and the increase in lactate was similar under Gen 3.0 and Gen 3.1 conditions as well as in the two media (complete medium and deterministic medium) used for reactivation amplification.

In some cases, the standard basal medium contains 2 mM L-glutamine and is supplemented with 2 mM GlutaMax ^™ to compensate for the natural degradation of L-glutamine into L-glutamic acid and ammonia under culture conditions.

In some cases, the specified (serum-free) culture medium used does not contain L-glutamine compared to the basal medium and is supplemented only with GlutaMax ^™ at a final concentration of 2 mM. GlutaMax ^™ is a dipeptide of L-alanine and L-glutamine, which is more stable than L-glutamine in aqueous solution and does not spontaneously degrade into glutamate and ammonia. Instead, the dipeptide gradually dissociates into individual amino acids, thereby maintaining a low but sufficient concentration of L-glutamine to sustain stable cell growth.

In some cases, the concentrations of glutamine and GlutaMax ^™ decreased slightly on scale-up days but increased on collection days, reaching levels similar to or closer to those on reactivation days. For L4064, the concentrations of glutamine and GlutaMax ^™ showed a slight decrease at a similar rate under different conditions throughout the process.

The ammonia concentration was higher in the samples grown in the standard medium containing 2 mM glutamine + 2 mM GlutaMax ^™ compared to the samples grown in the defined medium containing 2 mM GlutaMax ^™ . Furthermore, as expected, a gradual increase or accumulation of ammonia was observed during cultivation. No difference in ammonia concentration was observed under the three different test conditions.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats on L4063 and L4064 cells during the Gen 3 and Gen 3.1 processes was measured using Flow-FISH technology. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Telomere measurements were performed. Telomere lengths in the samples were compared with those in the control cell line (1301 leukemia). The control cell line was a tetraploid cell line with long, stable telomeres that allowed for the calculation of relative telomere lengths. Similar telomere lengths were observed in the Gen 3 and Gen 3.1 processes evaluated in both tumors.

TCR Vβ lineage analysis

To determine the clonal diversity of cell products generated in each process, TIL end products were analyzed for clonal diversity analysis by sequencing the CDR3 portion of the T cell receptor.

Compare three parameters across three conditions:

● Diversity index of unique CDR3 (uCDR3)

● Total uCDR3 percentage

●For the first 80% of uCDR3:

○ Compare the total percentage of uCDR3 copies

○ Compare the frequency of unique clones

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 975 sequences, which is equivalent to 88% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 2163 sequences, which is equivalent to 87% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

The number of unique CD3 sequences identified from 1 × ^10⁶ cells collected on day 16 for different processes. Based on the number of unique peptide CDRs within the sample, the Gen 3.1 test conditions showed slightly higher clonal diversity compared to Gen 3.0.

The Shannon entropy diversity index is a reliable and commonly used comparative measure because the Gen 3.1 condition showed slightly higher diversity in both tumors compared to the Gen 3 process, indicating that the TCRVβ lineage of the Gen 3.1 test condition was more polyclonal than that of the Gen 3.0 process.

Furthermore, for tumors L4063 and L4064, the TCR Vβ lineage under the Gen 3.1 test conditions showed more than 87% overlap with the corresponding lineage of the Gen 3.0 process.

On the reactivation day, the IL-2 concentration on the used medium for the Gen 3.1 test of L4064 was lower than expected (similar to the Gen 3.1 control and Gen 3.0 conditions).

The low value may be attributed to pipetting error, but since the sample size was very small, it was impossible to repeat the measurement.

Conclusion . Compared with the Gen 3.0 and Gen 3.1 controls, the Gen 3.1 test conditions, which included feeder cells and OKT-3 on day 0, showed a higher TVC at day 16 collection. The TVC of the final product under the Gen 3.1 test conditions was approximately 2.5 times higher than that under Gen 3.0.

For the two tumor samples tested, the Gen 3.1 test conditions, with the addition of OKT-3 and feeder cells on day 0, reached the maximum capacity of the culture flasks at collection. Under these conditions, if a maximum of four flasks were started on day 0, the final cell dose could be between 80 and 100 × ^10⁹ TILs.

Maintain all quality attributes, such as phenotypic characterization, including the purity, depletion, activation, and memory markers of the final TIL product, between the Gen 3.1 test and the Gen 3.0 process.

In both tumor types analyzed, the final TIL product IFN-γ production in Gen 3.1 with added feeder cells and OKT-3 on day 0 was 3-fold higher than that in Gen 3.0, indicating that the Gen 3.1 process produces efficient TIL products.

No differences in glucose or lactate levels were observed under any of the test conditions. No differences in glutamine and ammonia were observed between the Gen 3.0 and Gen 3.1 processes under any of the culture medium conditions. Lower glutamine levels in the culture medium did not limit cell growth, indicating that the addition of GlutaMax ^™ alone to the culture medium is sufficient to provide the nutrients required for cell proliferation.

Scaling up was performed on days 11 and 10, respectively, and no significant difference was observed in the number of cells reached on the process's collection day. Furthermore, metabolite consumption was similar throughout the process in both cases. These observations suggest that the Gen 3.0 optimized process offers flexibility in terms of processing days, thereby promoting flexibility in manufacturing schedules.

As measured by CDR3 TCRab sequence analysis, the Gen 3.1 process, which added feeder cells and OKT-3 on day 0, showed higher clonal diversity compared to Gen 3.0.

Figure 32 illustrates the implementation scheme of the Gen 3 process (Gen 3 optimization process). Standard medium and serum-free CTS Optimizer medium can be used for TIL amplification in the Gen 3 optimization process. When using serum-free CTS Optimizer medium, it is recommended to increase the final concentration of GlutaMax ^™ on the medium to 4 mM.

Example 11: PD-1 gene knockout in TIL using TALEN

The embodiment described above illustrates the use of TALEN to generate PD-1 gene knockout (KO) TILs. In this embodiment, the general procedure is as follows:

Day 0 - Pre-REP

Day 7 - Activation

Day 12 - Electroporation

Day 13 - REP

Day 18 - Scale Expansion

Day 22 - Collection

Overview : This study used genetic engineering techniques to permanently knock out the PDCD-1 gene (PD-1 surface protein) using an mRNA construct. PD-1KO TILs were generated from one lung (L4256) and two melanoma (M1207 and M1209) tumors and then characterized.

Background : Binding between immune checkpoint receptors and their ligands has been shown to suppress T cell function. PD-1, a key inhibitory regulator expressed by activated/depleted T cells, has been extensively studied and is a major target for immunotherapy. Given the success of anti-PD-1 therapy in improving clinical outcomes in cancer patients, approaches targeting the PD-1/PD-L1 axis could enhance the efficiency of immunotherapy. Adoptive TIL therapy is a salvage immunotherapy for cancer, with an objective response rate of 56% in melanoma patients. Since PD-1 is regulated in TILs in response to antigen stimulation in vivo, the interaction between PD-1 and PD-L1 expressed by tumor cells may suppress TIL function. Gene editing of PD-1 has demonstrated high specificity for permanent gene disruption in human and mouse T cells. In the B16 mouse model, adoptive transfer of PD-1 T cells revealed superior tumor control and enhanced effector function and proliferative capacity of tumor-site infiltrating T cells. Therefore, this study examines whether PD-1 KO-mediated TILs can enhance TIL function in vivo after adoptive transfer.

Experimental design : Clinical manufacturing scale operations were performed as shown in the experimental layout in Figure 34, and an overview of the clinical manufacturing PD-1KO TIL process is provided in Tables 42 and 43.

The method is also applicable to the generation of PD-1/CTLA-4 dual-gene knockout TILs, PD-1/LAG-3 dual-gene knockout TILs, PD-1/CISH dual-gene knockout TILs, PD-1/TIGIT dual-gene knockout TILs, and PD-1/CBL-B dual-gene knockout TILs. In such cases, during the electroporation step, dual-gene knockout TILs can be generated by electroporating TILs with PD-1 and CTLA-4 TALEN mRNA, PD-1 and LAG-3 TALEN mRNA, PD-1 and CISH TALEN mRNA, PD-1 and TIGIT TALEN mRNA, or PD-1 and CBL-B TALEN mRNA, respectively.

Table 42: Outline of Pre-REP, Activation, Electroporation, and Resting Processes (Part I)

Table 43: Outline of REP, Scale-up, and Collection Process (Part II)

Results : Table 44 specifies the acceptance criteria used to evaluate the full-scale experimental efficacy, Table 45 specifies the additional end-product characterization tests performed, and Table 46 lists the tumors and related histories used in this study.

Table 44: In-process and Collected Product Release Testing and Acceptance Standards

Table 45: Characterization of the final product

Table 46: Tumors used in this study

On day 5, the total live cell count (TVC) after pre-REP yielded an average of 45 × ^10⁶ cells, with a mean survival rate of 73%. Cell doubling after activation was consistent across all three tumor types. Melanoma and the cervical indication both showed similar activation status, with activation markers of >50% CD25+ and CD71+. However, the lung tumor L4213 showed lower CD71 expression, at 33%. Ki67 expression was consistently greater than 90% in all three samples, indicating active cell proliferation. Table 47 summarizes the pre-REP and post-activation output for the three tumor types used in the study.

Table 47: Summary of Pre-REP and Post-Activation Cell Culture

parameter	Acceptance Standards	L4256	M1207	M1209
					Number of segments	≤100 segments	73	40	95
<![CDATA[TVC(×10<sup>6</sup>)]]>	≥5	26.9	13.4	69.2
					Survival rate (%)	N/A	97.9	88.6	97.5
<![CDATA[TVC(×10<sup>6</sup>)]]>	N/A	120.0	37.8	419.0
					Survival rate (%)	N/A	92.0	82.6	92.8
Double (D7 to D12)	N/A	2.2	1.5	2.6
					CD45+ / CD3+%	N/A	80.4	83.1	60.1
<![CDATA[CD25+%<sup>1</sup>]]>	N/A	92.2	88.5	66.8
					<![CDATA[CD71+%<sup>1</sup>]]>	N/A	75.6	87.8	48.9
<![CDATA[Ki-67+%<sup>2</sup>]]>	N/A	78.6	81.0	7.3

^1. Gating strategy of the group after activation on day 10: Lymphocytes > SSC single peak > FSC single peak > Live cells > CD3+CD45 > CD25+/CD71+

^2. Ki67 group's gating strategy: Lymphocytes > SSC unimodal > FSC unimodal > Live cells > CD45+ > Ki67+

On day 13 (after rest), survival rate and TVC recovery rate were calculated to determine the efficiency of electroporation. For all conditions, the recovery rate of the percentage of cells surviving after electroporation ranged from 82% to 92%. In electroporated samples, the TVC recovery rate ranged from 16% to 30%. Data from electroporation and resting are summarized in Table 48.

Table 48: Summary of Electroporation and REP Initiation

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The recovery rate is calculated by dividing the TVC or survival rate after electroporation by the TVC or survival rate before electroporation and then multiplying by 100.

^2. The TVCs injected into the REP were the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

The same number of cells were used to initiate the REP under both experimental and control conditions. As the REP scaled up, the average cell doubling occurred 4 times under all experimental and control conditions. At REP collection, TVC yield in the TriLink experimental group was similar to that in the simulated control group (TVC difference ≤20%). Non-electroporated TVC yield was slightly higher than that in the TriLink and simulated control groups. An average of 9.8 cell doublings was observed between day 11 and day 20.

The final product doses of the TriLink group were 16.5, 28.5, and 28.7 × ^10⁹ , respectively, with >90% cell viability and >98% CD45+CD3+ cell count. The final product was a highly enriched TIL product.

The data from the REP initiation and collection are summarized in Table 49.

Table 49: Post-REP Summary

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The TVC inoculated into the REP was the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

^2. The recovery rate is calculated by dividing the TVC after LOVO by the TVC before LOVO and multiplying by 100.

^3. Assuming all cells proceed to the hypothetical yield in both experimental and control conditions. On day 12 (before electroporation), cells are allocated for experimental (TriLink mRNA) and control conditions (simulated). Hypothetical yield is calculated by multiplying the collection by 3.

^4. Cell doubling is calculated using the formula “＝LOG(TVC on day 22 / TVC on day 11) /LOG(2)”.

^5. LOVO did not follow the plan.

^{The six} batches of materials were small in scale and did not undergo LOVO testing.

All TriLink mRNAs met the acceptance criteria.

Data on the gene knockout efficiency of PD-1KO TIL products evaluated are summarized in Table 50.

Table 50: TIL PD-1KO Summary

Data evaluating the TIL function of the collected samples (including IFNg, granzyme-B, and CD107a release output) are summarized in Table 51.

Table 51: Summary of TIL Functions

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. Stimulation gating targeting live cells/CD3+

The functionality of TILs was characterized based on overnight stimulation of the final product with anti-CD3/anti-CD28/anti-CD137 Dynabeads. Supernatants were collected and frozen 24 hours after stimulation. ELISA was performed to assess the concentrations of IFNg and granzyme B released into the supernatant. IFNg release was greater than 500 pg/mL, and all TIL cultures secreted high levels (15036 pg/mL–40185 pg/mL) of granzyme B after stimulation.

CD107a (LAMP-1) is a marker of lymphocyte degranulation and is associated with cytotoxic activity following antigen or polyclonal stimulation (Aktas E et al., 2009). Flow cytometry was used to analyze the cell surface expression of CD107a in response to PMA stimulation for 2 hours in both CD4 ⁺ and CD8 ⁺ cells. The results presented in Table 51 compare CD107A expression measurements with those under unstimulated conditions. Similar to the TIL products generated in previous studies, most of the TILs in the final product expressed CD107A upon stimulation with PMA/IO (CD4+ and CD8+ TILs).

Multicolor flow cytometry was used to characterize TIL purity, identity, and the memory subset of REP TILs. ≤1% of detectable B cells, monocytes, or NK cells were present in the final collected TILs (Table 11). REP TILs were primarily composed of TCRa/b cells with effector memory differentiation. The CD4/CD8 ratio varied between samples but was consistent across different conditions within a single sample. No significant differences were observed between experimental and control conditions.

Data showing TIL purity, identity, and memory phenotype are summarized in Table 52.

Table 52: Characterization of TIL purity, identity, and memory phenotype

Note: The gating strategy for TIL purity is as follows:

Monocytes: viable cells%, CD14+

NK (Natural Killer) cells: viable percentage, CD14-, CD3-, CD56+CD16+

B cells: viable percentage, CD14-, CD3-, CD19+

T-CM-Central Memory T Cells

T-EM-effect memory T cells

T-EFF/TEMRA-effect memory and RA+ effect memory

Data showing TIL activation and TIL depletion levels for CD4+, CD8+, and CD3+ are summarized in Tables 53-55.

Table 53: Activation and Depletion States of CD4+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD4+ > CD4+ frequency %

Table 54: Activation and Exhaustion States of CD8+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD8+ > CD8+ frequency %

The CD4 and CD8 differentiation, activation, and depletion states under the TriLink mRNA condition were comparable to those under the control condition.

Table 55: Activation and Depletion States of CD3+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD3+ frequency %

The activation and depletion status of TriLink mRNA in the CD3+ population were comparable to those in the control population.

Conclusion : The PD-1KO TIL process was developed at clinical manufacturing scale to amplify PD-1KO TIL to >16e9 within 20 days. All three batches of TriLink mRNA used at development scale met the acceptance criteria for release parameters.

All quality properties of TILs generated using the PD-1-KO TIL process, such as phenotypic characterization (including purity, memory, activation, exhaustion markers, and function), are comparable to those of melanoma Gen 2. See Table 56.

In summary, this work demonstrates that greater than 50% PD-1KO efficiency can be achieved in the clinical manufacturing of PD-1KO TILs using TriLink mRNA, thus supporting the use of TriLink mRNA in clinical manufacturing.

Table 56: Summary Table

The alternative PD-1 gene knockout (KO) TIL process is shown in Figure 35 and Table 57.

Table 57: Overview of the alternative full-scale process for PD-1 TIL culture

Example 12: Preclinical activity and manufacturing feasibility of gene-modified PDCD-1 gene knockout (KO) tumor-infiltrating lymphocyte (TIL) cell therapy

background

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TILs; lifileucel, LN-145) has demonstrated encouraging efficacy and safety in both ICI-injected and ICI-naïve settings in patients with advanced solid tumors. A single-dose lifileucel TIL cell therapy achieved a durable response in a post-ICI setting in patients with advanced/unresectable melanoma (Sarnaik AA, Hamid A, Khushalani NI et al., J Clin Oncol. 2021; 39(24):2656-2666; Larkin JMG, Sarnaik A, Chesney JA et al., J Clin Oncol. 2021; 39(Supplement 15): Abstract 9505.), with an objective response rate (ORR) of 36%, and the duration of response (DOR) was not reached after 33.1 months of follow-up. In patients with advanced melanoma who have not received ICI treatment, early combination therapy with lifileucel and pembrolizumab resulted in an ORR of 60% and a complete response rate of 30% (O'Malley D, Lee S, Psyrri A et al., J Immunother Cancer. 2021; 9(Supplement 2): Abstract 492).

Despite the effectiveness of anti-programmed cell death protein (PD)-1 ICI therapy, it is limited by poor tumor penetration, internalization, and endocytic clearance (Thurber GM, Schmidt MM, Dane Wittrup K. Adv Drug Deliv Rev. 2008; 60(12):1421-1434; Less JR, Posner MC, Boucher Y, Borochovitz D, Wolmark N, Jain RK. Cancer Res. 1992; 52(22):6371-6374; Netti PA, Baxter LT, Boucher Y, Skalak R, Jain RK. Cancer Res. 1995: 55(22): 5451-5458; Bordeau BM, Balthasar JP. Cancer Biol Med. 2021; 18(3): 649-664; Saga T, Neumann RD, Heya T et al., Proc Natl Acad Sci U SA. 1995; 92(19): 8999-9003); In contrast, TILs are actively transported into the tumor, can move independently by chemitraction, and are not cleared or internalized (Restifo NP, Dudley ME, Rosenberg SA. Nat Rev Immunol. 2018; 12(4): 269-281). Administration of anti-PD-1 antibodies is associated with immune-related adverse events (AEs) due to nonspecific upregulation of immune pathways via systemic activation and proliferation of T cells (Postow MA, Sildow R, Hellmann MD. N Engl J Med. 2018; 378:158-168).

IOV-4001 is a PDCD-1 gene knockout (KO) TIL cell therapy that enhances the efficacy of TIL cell therapy and eliminates the need for systemic anti-PD-1 therapy, while avoiding short-term and long-term systemic adverse events associated with anti-PD-1/PD-L1 therapy. Transcription activator-like effector endonucleases (TALENs) are hybrid molecules composed of a DNA-binding domain and a FokI nuclease. Combination of the two TALEN arms of the PDCD-1 gene encoding PD-1 mediates DNA double-strand breaks, leading to gene damage and PD-1 inactivation (Gautron AS, Juillerat A, Guyot V et al., Mol Ther Nucleic Acids. 2017; 9:312-321; Menger L, Sledzinska A, Bergerhoff K et al., Cancer Res. 2016; 76:2087-2093; Qasim W, Zhan H, Samarasinghe S et al., Sci Transl Med. 2017; 9(374):eaaj2013). A process has been established to generate TALEN-mediated PDCD-1KO TILs and amplify them to a treatment-relevant number with potent effector function and phenotypic markers indicating functional TILs (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; Sep 19–21, 2020. Abstract 1052p).

This embodiment describes the (1) preclinical activity of IOV-4001 in vivo, (2) clinical-scale manufacturing process development, and (3) phenotypic and functional characterization.

method

In summary, hIL-2NOG mice transplanted with melanoma tumor cells received autologous PD-1KO TILs (generated using an mRNA construct), TIL-mimicking (electroporation without TALEN), TIL-mimicking plus anti-PD-1 antibody adoptive transfer, or no adoptive transfer (n=14 each). Tumor size was measured twice weekly for 39 days. A 22-day clinical-scale manufacturing process was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, for the generation of PD-1KO TILs. The final PD-1KO TIL product was characterized by total live cells (TVC), purity (survival percentage), identity (CD45 ⁺ CD3 ⁺ %), and function (PD-1KO efficiency).

manufacture :

A 22-day clinical-scale manufacturing process for IOV-4001 was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, to generate PDCD-1KO TILs (using an mRNA construct). Development operations were performed at Iovance Process Development (Tampa, FL) on a non-Good Manufacturing Practice (GMP) scale. Manufacturing operations were performed at a contract manufacturing organization (CMO) on a GMP scale.

In vivo antitumor activity :

Mice expressing human interleukin-2 (hIL2) under the control of the cytomegalovirus promoter (hIL-2NOG)15 were transplanted with melanoma tumor cells and received (A) autologous PDCD-1KO TIL, (B) TIL mimicry (TIL electroporation without TALEN), (C) TIL mimicry plus anti-PD-1 antibody, or (D) TIL-free adoptive transfer (n=14 each). Tumor size was measured twice a week for 39 days.

Product release :

The final PDCD-1KO TIL product was characterized as follows:

●Total viable cells (TVC) and purity (percentage of viable cells) were determined using an NC-200 ^™ (ChemoMetec, Denmark) automated cell counterstaining system via acridine orange/4',6-diamidindo-2-phenylindole (DAPI) counterstaining.

● Identity (CD45+CD3+ phenotype), analyzed by immunofluorescence staining and flow cytometry.

●Efficacy (interferon-γ [IFNγ] release), measured by ELISA using an IFNγ ELISA kit (R&D Systems, Minneapolis, MN, USA).

PDCD-1KO efficiency was evaluated by flow cytometry based on PD-1 expression in PDCD-1KO TILs compared with mimic TILs.

Phenotype :

Two multicolor flow cytometry teams were used to determine extended phenotypic markers of the final collected PDCD-1KO TIL products to characterize TIL purity, identity, memory subsets, activation, and depletion states.

Characterization :

IL-2 assay: To assess the safety of the PDCD-1KO TIL product, the in vitro proliferation capacity of the final product in the absence of IL-2 was evaluated over a 28-day period.

Karyotype determination: Cytogenetic examination was performed by NeoGenomics Laboratories (Fort Myers, FL, USA). Briefly, cryopreserved PDCD-1KO TIL samples were quiescent and activated to collect metaphase cells for G-banding cytogenetic analysis. Three replicates of mitotic cells were analyzed, fixed, and stained for G-banding.

Statistical analysis :

Unpaired Student's t-test was used to analyze phenotypic differences, and Wilcoxon's rank-sum test was used to detect differences in in vivo studies in mice; p < 0.05 was considered statistically significant.

result

In summary, for mice treated with PD-1KO TIL (6±2.8), mean ± SEM tumor size ( ^mm² ) at day 39 showed superior tumor control compared to simulated TIL (26±8.5, P<0.05), simulated TIL + anti-PD-1 (33±8.8, P<0.01), and no adoptive TIL metastasis (112±8.4, P<0.0001). Product properties from six clinical-scale manufacturing operations of PD-1KO TIL were acceptable, with median (range) TVC, purity, and identity of 8.3 × ^10⁹ (0.9 × ^10⁹ –35.7 × ^10⁹ ), 94% (91%–99%), and 99% (98%–99%), respectively. The median (range) PD-1KO efficiency was 48% (31%–84%). PD-1KO TILs exhibit comparable functions and phenotypes (differentiation, memory, activation, and exhaustion) to those of analog TILs.

As shown in Figure 38, enhanced in vivo antitumor activity was observed in mice treated with PDCD-1KO TIL compared to mice treated with TIL-mimicking alone or TIL-mimicking alone plus anti-PD-1 antibody.

As shown in Figure 39, all PDCD-1KO TIL products met the release criteria for dose, purity, identity, potency, and PDCD-1KO efficiency. No statistically significant differences were observed between the development and manufacturing processes. Both development and manufacturing processes produced final PDCD-1KO TIL products with comparable doses (A) and survival rates (B). The median (range) identity (CD45+CD3+%) of the final TIL products in the development and manufacturing processes was 98.5% (98%–100%) and 98.7% (96%–99%), respectively (C). The median IFNγ release in the development and manufacturing processes of the final PDCD-1KO TIL products was 4015 pg/mL and 4725 pg/mL, respectively (D). The median (range) PDCD-1KO efficiencies in the development and manufacturing processes were 63% (48%–81%) and 62% (31%–91%), respectively (E). The PDCD-1KO TIL product is comparable to the simulated TIL in terms of growth, purity, identity, and potency. As previously shown (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; September 19–21, 2020. Abstract 1052p), the dose, purity, identity, and potency results between the simulated and PDCD-1KO were comparable in development operations (data not shown).

As shown in Figure 40, the CD28 biomarker was highly expressed in both PDCD-1KO and simulated TILs in the development and manufacturing samples, while other biomarkers such as CD27, CD57, and KLRG1 were expressed at low levels (A). Subsets of naive (TN), central memory (TCM), effector memory (TEM), and effector memory RA+ (TEMRA) T cells were defined using CD45RA and CCR7 expression. Most TIL batches primarily exhibited the effector memory phenotype (B). No statistically significant differences in TIL differentiation biomarkers or memory phenotypes were observed between PDCD-1KO and simulated TILs during the development and manufacturing operations.

As shown in Figure 41, multicolor flow cytometry was used to characterize TIL activation and inhibitory receptor expression on CD4+ (A) and CD8+ TILs (B). No statistically significant differences in biomarker expression were observed between PDCD-1KO and simulated TILs during development and manufacturing operations.

As shown in Figure 42, the IL-2-independent proliferation assay demonstrated that no PDCD-1KO TIL products underwent malignant transformation after TALEN-mediated genome editing. In the absence of IL-2 (A and B), neither stimulated (anti-CD3/CD28) nor unstimulated samples proliferated. All samples cultured in the presence of IL-2 showed proliferation (A and B).

The karyotype analysis results from the PDCD-1KO TIL products are summarized in Figure 43. No clonal chromosomal abnormalities were observed in the G-banding analysis, indicating no genotoxicity following TALEN-mediated PDCD-1 genome editing. All samples analyzed by G-banding generated sufficient metaphase cells for a comprehensive study; normal G-banding patterns were observed.

in conclusion

In the presence or absence of anti-PD-1, PDCD-1KO TILs exhibited superior in vivo antitumor activity compared to mimic TILs (electroporation without TALEN), indicating that endogenous PD-1 inhibition can confer a functional advantage over antibody combinations. Clinical-scale manufacturing of PDCD-1KO TILs is feasible, and the quality properties and phenotype of the TIL products are acceptable. TIL properties were comparable across all development and manufacturing operations and met product release criteria. As determined by IL-2-independent proliferation assays, no malignant transformation occurred in PDCD-1KO TIL products following TALEN-mediated genome editing. No TALEN-induced clonal chromosomal abnormalities were identified by G-banding. Importantly, the absence of intact PDCD-1KO in the TIL products preserved other PD-1-dependent in vivo cellular functions. In conclusion, these data support the clinical investigation of IOV-4001 (an autologous PDCD-1KO TIL cell therapy) as a potential treatment option for patients with advanced solid tumors.

Example 13: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated, electroporated, and subjected to an 11-day REP process.

Pre-REP cells were stimulated for two days with plate-bound OKT3 (300 ng/ml) or TransAct (1:100), followed by electroporation of 5e6 cells resuspended in an electroporation buffer called “T buffer” in 4 mm spaced electroporation reaction cuvettes, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

After electroporation, cells were allowed to rest overnight at 30°C in CM1 medium containing IL-2, followed by REP. Simulated cells were seeded at different initial numbers (100kJ, 50kJ, 20kJ, 10kJ) and tests were performed to compare gene knockout efficiency.

result

Figures 44A and 44B show cell viability and recovery folds before electroporation. Specifically, Figure 44A shows cell viability after thawing compared to 2 days after stimulation with OKT3 or TransAct, and Figure 44B shows the recovery folds after thawing and stimulation with OKT3 or TransAct.

Figures 45A and 45B show the cell viability and recovery fold of cells after electroporation. Specifically, Figure 45A shows the recovery fold of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C, and Figure 45B shows the cell viability of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C.

Figures 46A-46C show that, in the case of CD3+, CD8+, and CD4+ cells, TILs stimulated by TransAct exhibited lower KO efficiency compared to TILs stimulated by OKT3.

Example 14: Comparison of overnight rest at 30°C and 37°C

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated and electroporated.

Pre-REP cells were stimulated with TransAct (1:100) for two days, followed by electroporation of 5e6 cells resuspended in T buffer in electroporation reaction cups with a 4 mm spacing, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

Following electroporation, cells were seeded in equal numbers into 96-well plates and allowed to rest overnight at 30°C or 37°C in CM1 medium containing IL-2 or IL-15. Cells were then counted to determine the fold resuscitation and viability.

result

Figures 47A and 47B show cell viability and recovery folds after electroporation under all conditions (IL-2 or IL-15). Specifically, Figure 47A shows the recovery folds of cells after overnight rest at 30°C or 37°C, and Figure 47B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 48A and 48B show the cell viability and fold recovery rate of cells after electroporation when 6000 IU/mL IL-2 was used. Specifically, Figure 48A shows the fold recovery rate of cells after overnight rest at 30°C or 37°C, and Figure 48B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 49A and 49B show cell viability and recovery folds after electroporation under various conditions. Specifically, Figure 49A shows the recovery folds of cells after overnight rest at 30°C or 37°C when using various concentrations of IL-2 or IL-15, and Figure 49B shows the cell viability after overnight rest at 30°C or 37°C when using different concentrations of IL-2 or IL-15.

Gene knockout efficiency was evaluated as described in Example 13. TILs were stimulated overnight with anti-CD3/CD28 beads at a ratio of 1 bead to 5 cells. Figures 50A-50C show that a decrease in KO efficiency was observed at the recommended 1:100 dilution under non-GMP TransAct stimulation.

Example 15: Comparison of TALEN mRNA concentration and incubation conditions

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed, activated, electroporated and subjected to a 10-day REP process.

Pre-REP cells were stimulated with OKT3 (300 ng/mL plate-bound) for two days, followed by electroporation of 1e6 cells resuspended in T buffer in electroporation reaction cuvettes with a 1 mm spacing. The right arm or left arm was given 4 μg, 2 μg, 1 μg, and 0.5 μg of PD-1 TALEN per 1e6 cells, respectively.

After electroporation, cells were seeded in the same number in 48-well plates and allowed to rest overnight in CM1 medium containing IL-2 at 30°C or 37°C, followed by REP.

result

Figures 51 and 52 show the cell viability and fold recovery rate of cells after electroporation with different concentrations of PD-1 TALEN mRNA. Specifically, Figure 51 shows the cell viability after electroporation with different concentrations of PD-1 TALEN mRNA, and Figure 52 shows the fold recovery rate after electroporation with different concentrations of PD-1 TALEN mRNA. No significant changes in viability or recovery rate were observed after electroporation with different concentrations of PD-1 TALEN mRNA.

Figures 53A and 53B show the cell viability and fold recovery rate when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C. Specifically, Figure 53A shows the fold recovery rate of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C, and Figure 53B shows the cell viability of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C. No significant changes in viability or recovery rate were observed when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C.

Gene knockout efficiency was evaluated as described in Example 13. Figures 54A-54C show the PD-1 KO efficiency in CD3+, CD8+, and CD4+ cells. The optimal KO efficiency of PD-1 TALEN was observed at 4 μg mRNA/million cells and overnight rest at 30°C. This concentration can be target-specific and also depends on the cell concentration used during electroporation. The concentration of TALEN mRNA used during the electroporation process did not appear to affect recovery rate and survival.

Example 16: Comparison of washing steps and centrifugation speed

method

Pre-REP TIL lines (N=4) from different indications (head and neck and lung) were thawed, activated (OKT3 plate binding 300 ng/mL), and subjected to washing steps that are usually performed before electroporation.

Pre-REP cells were stimulated for two days and then aliquoted into individual tubes at 5e6 cells per tube. They were washed with PBS, precipitated, washed with cell perforation buffer, precipitated again, and then resuspended in cell perforation buffer. Cells were resuspended in 500 μL of cell perforation buffer and counted by diluting the cells 1:10 in CM1. Cell counting was performed after each rotation to determine the percentage of cells lost during the washing step.

Repeat the procedure using different centrifugation speeds (400g, 300g, 200g, for 5 minutes) to improve cell recovery rate.

result

Figures 55A and 55B show the cell number (Figure 55A) and viability (Figure 55B) after various washing steps involving centrifugation at 400g for 5 minutes and rotation 3 times.

Figures 56A and 56B show the number of cells after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 57A and 57B show cell viability after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 58A and 58B show the total number of cells and the total survival rate of cells after various rotation conditions, and Figure 59 shows the percentage of cells lost after various rotation conditions.

The results showed that washing with cell perforation buffer led to cell loss, and that the recovery rate may have been improved when cells were centrifuged in cell perforation buffer at 300g instead of 400g. Therefore, a single cell perforation buffer washing step is preferred.

Example 17: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates containing 300 ng/mL, TransAct 1:10 and TransAct 1:17.5, and then electroporated with PD-1 TAL.

result

Figures 60A-60C show the percentage loss and survival during electroporation using various stimulation methods, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation (Figure 60C). The results indicate that TransAct stimulation exhibits better recovery and survival rates after electroporation compared to OKT3.

Figures 61A-61C show the PD-1 gene knockout efficiency in CD3+ (Figure 61A), CD4+ (Figure 61B), and CD8+ (Figure 61C) cells using various stimulation methods. The results indicate that activation with OKT3 and TransAct leads to similar KO efficiency.

Figures 62A-62B show the cell viability (Figure 62A) and fold expansion (Figure 62B) of REP collections using various stimulation methods.

Example 18: Comparison of incubation temperatures after electroporation

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates at 300 ng/mL, and then electroporated with PD-1 TAL.

After electroporation, cells were incubated overnight at different temperatures (25, 28, 30, 32, 35, 37 °C) and REP was performed the following day. Cell loss and viability, as well as PD-1 gene knockout efficiency, were evaluated after overnight incubation.

result

Figures 63A-63B show the percentage of cell loss (Figure 63A) and cell viability (Figure 63B) after electroporation at different incubation temperatures.

Figures 64A-64C show the gene knockout efficiency in CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells using different incubation temperatures.

Figures 65A-65B show the fold expansion (Figure 65A) and cell viability (Figure 65B) of REP collections at different incubation temperatures.

Example 19: Comparison of Stimulus Day Timing Selection

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated with GMP TransAct 1:17.5 on different dates (days 0, 3, 5, and 7).

Electroporation of cells with PD-1 TAL was performed on days 9 and 12 to determine the optimal dates for stimulating pre-REP cells to maximize PD-1 KO efficiency and pre-REP cell number.

result

Figures 66A-66C show cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C) of cells stimulated on different dates.

Figure 67 shows the percentage growth of cells stimulated on different dates at 3 days of rest.

Example 20: Overview of PD-1KO TIL Products

"PD-1KO TIL Product" is an autologous tumor-infiltrating lymphocyte (TIL) preparation. These lymphocytes have been genome-edited with a transcription activator-like effector nuclease (TALEN) to disrupt the gene for programmed cell death protein-1 (PD-1). The PD-1 TALEN is engineered to bind to a distinct DNA sequence adjacent to the nucleotide lysis target site on the PDCD1 gene. DNA binding leads to the dimerization of two separate FokI endonuclease domains, producing a functional DNA endonuclease that can generate a sequence-specific double-strand break (DSB) in exon 2 (the central protein-coding region of PDCD1). This DSB is repaired via endogenous error-prone DNA repair pathways (e.g., non-homologous end joining (NHEJ)), resulting in nucleotide insertions or deletions, thereby disrupting the protein-coding sequence of PD-1.

The 4-step PD-1KO TIL product manufacturing process is a modified version of the method used to produce the autologous TIL product lifileucel (IND 16317 and 16819). This process consists of the following steps: a first step of TIL growth in the presence of IL-2, followed by transient activation of T cells with TransAct (a bead-free colloidal polymer nanomatrix conjugated with humanized CD3 and CD28 agonists), electroporation of PD-1KO TALEN mRNA (left and right arms), overnight rest at 30°C, and a final rapid expansion protocol (REP) step.

Cross-study PD-1 KO TIL product characterization data (Table 58) and process development data indicate that changes in the manufacturing process do not affect product quality (characteristics, survival rate, PD-1 gene knockout efficiency) and result in an average PD-1 KO efficiency of approximately 60%. In the PDX mouse model system, its antitumor activity is superior to that of the simulated control TIL in the presence of pembrolizumab.

Table 58. List of Non-Clinical Studies

Abbreviations: GLP = Good Laboratory Practice; IL-2 = Interleukin-2; KO = Gene Knockout; OCA = Oligonucleotide Capture Assay; PD-1 = Programmed Cell Death Protein-1; PDCD1 = Gene of Programmed Cell Death Protein-1; PDX = Patient-Derived Xenograft; TALEN = Transcription Activator-like Effector Nuclease; TCR = T Cell Receptor; TIL = Tumor-Infiltrating Lymphocytes

¹ This study evaluates different batches of PD-1KO TIL products, some of which were evaluated as part of a GLP compliance study.

Overview

The non-clinical studies conducted support the safety of the PD-1KO TIL product initiating clinical studies through the following:

● Confirmed that PD-1 TALEN expression in TILs is transient after electroporation;

● Characterization using NGS confirmed that 19 of the top 20 potential off-target sites identified in the OCA analysis were free of TALEN-related mutagenesis. Minimal off-target activity was detected at one candidate site (Cand 3). This is unlikely to pose a safety risk to the product because of the low mutation frequency and the fact that the site corresponds to a non-coding RNA locus not known to be associated with the disease.

● No clonal chromosomal aberrations were identified in the karyotype analysis of the PD-1KO TIL drug product, therefore there is no evidence that TALEN-mediated genome editing has significant genotoxicity;

● No evidence of IL-2-independent proliferation was found, thus demonstrating that no malignant transformation was observed in any of the PD-1KO TIL drug products tested.

Furthermore, non-clinical studies support the potential antitumor activity and mechanism of action of PD-1KO TIL products based on the following observations:

● Reproducible high levels of TALEN-mediated PDCD1 mutagenesis (approximately 60% on average) were achieved in the genome of autologous TILs, with the PDCD1 mutagenesis rate correlated with decreased PD-1 expression. Therefore, PD-1KO TIL products constitute true PD-1KO gene therapy products.

● The general characteristics of autologous TILs, which are considered crucial for anti-tumor activity, have been shown to be unaffected or positively affected by PD-1KO, including survival, proliferative capacity, differentiation status, memory T cell subset, activation/exhaustion status, TCR Vβ diversity, T cell effector function, and cell lysis activity.

● Enhanced tumor burden control was observed in PDX mouse models treated with ACT and PD-1KO TIL products, compared with control groups that did not use ACT, ACT with unmodified autologous TIL, and ACT with unmodified autologous TIL combined with anti-PD-1 antibody.

In summary, these studies demonstrate that isolating, expanding, and genetically engineering PD-1 knockout T cells from various tumors is safe and exhibits potent antitumor activity in both in vitro and in vivo models. These observations suggest that PD-1KOTIL products possess the potential and manageable safety profile of an autoimmune therapy for treating patient populations constituting unmet medical needs.

result

Figures 68A-68C show the PD-1 gene knockout efficiency. Figure 68A shows the PD-1 KO efficiency determined by measuring PD-1 receptor expression on the PD-1 KO TIL product using flow cytometry. Figure 68B shows the PD-1 KO efficiency determined by measuring the total number of PDCD1 genes with insertions/deletions using NGS. Figure 68C shows the correlation between the PD-1 KO efficiency assessed by flow cytometry and the PDCD1 gene modifications determined using NGS.

Figure 69 shows that most of the insertions/deletions in PDCD1 after TALEN genome editing were nucleotide deletions.

Phenotypic characterization

The proliferative capacity, survival, and immunophenotype of PD-1KO TIL products relative to unmodified autologous TILs were evaluated using a study-scale process derived from five different types of tumors (breast (n=5), lung (n=2), HNSCC (n=2), melanoma (n=2), and ovary (n=1)).

Cell viability and proliferation capacity

The PD-1KO TIL product exhibited cell viability comparable to non-electroporated (NE) TILs, with an average viability of approximately 93% for both types of samples. The proliferative capacity of the PD-1KO TIL product was also comparable to that of NE TILs, with average fold increases of 1217 and 1565, respectively, during the REP step of the production process.

Differentiation status of PD-1KO TIL products and NE TIL

PD-1KO TILs exhibit a differentiation state comparable to NE TILs, as determined by similar expression levels of CD27, CD28, CD56, BTLA, and KLRG-1. Therefore, PD-1 inactivation does not alter the differentiation state of TILs.

Memory T cell subsets in PD-1KO TIL products and NE TIL

In this study, over 98% of PD-1KO TIL products and NE TILs were T effector cells (CD45RA-CCR7-), while _{the TCM} subset (CD45RA-CCR7+) accounted for less than 1% of the cells analyzed. Importantly, the proportion of terminally differentiated T cells was negligible.

Activation and depletion states in PD-1KO TIL products and NE TIL

As indicated by the expression levels of CD69, CD25, and DNAM, the activation state of the PD-1KO TIL product was relatively high and comparable to that of NE TIL. Consistent with its recent activation, both the PD-1KO TIL product and NE TIL expressed high levels of co-inhibitory molecules (TIGIT and TIM-3). These results suggest that the activation and depletion states are not altered by PD-1 inactivation.

In summary, PD-1 gene knockout did not significantly affect TIL survival, proliferation capacity, or immune phenotype, indicating that the activity of PD-1KO TIL products associated with these biological characteristics was similar to that observed in unmodified autologous TILs.

Figures 70A-70B show the distribution of TCR Vβ isotypes in the CD3+PD-1- subset within the main PD-1KO TIL products and NE TILs. Main TILs generated from lung tumor L4022 (Figure 70A) and breast tumor EP11017 (Figure 70B) were electroporated with PD-1 TALEN mRNA (PD-1KO) or not electroporated (NE). Twenty-four TCR Vβ families were evaluated by flow cytometry in the CD3+PD-1-T cell subset. The percentage of TCR Vβ isotypes is plotted in pie charts. All remaining isotypes not identified by flow cytometry are pooled and shown in light blue (other). The color represents each TCR Vβ isotype, as indicated in the legend below the pie chart.

Figures 71A-71B show the effector function of PD-1KO TILs as measured by MLR and multifunctionality. For Figure 71A, TILs were generated in duplicate from melanoma (n=3), breast cancer (n=1), and lung cancer (n=1) samples. The responsiveness of NE TILs (black circles) and PD-1KO TILs (white circles) to HLA-mismatched lymphocytes was tested. IFN-γ secretion from the supernatant was assessed using ELISA. The horizontal and vertical bars represent the mean and standard error, respectively. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance. For Figure 71B, TILs were generated from melanoma (n=1) and breast cancer (n=3) samples. The multifunctionality intensity index (PSI), a measure of the ability to produce multiple cytokines at the single-cell level, was measured in activated host PD-1KO and NE TILs. The results are shown as a bar graph of the mean and corresponding standard error of cytokines involved in the inflammatory (pink), regulatory (beige), chemoattractive (purple), stimulative (blue), and effector (green) pathways. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance.

In vivo studies: Patient-derived xenograft (PDX) mouse model studies

This study compared the antitumor activity of PD-1KO TIL products with unmodified TILs in a patient-derived xenograft (PDX) model grown in hIL-2 transgenic mice. Paired autologous PDX tumor cell lines/TIL formulations derived from freshly excised human melanoma lesions (n=1; M1152) were used in this study. The PD-1KO efficiency level was 75%. No differences in cell viability, proliferation, differentiation, memory T cell subsets, or exhaustion status were observed between M1152 PD-1KO and simulated TILs.

Characterization of PDX-derived melanoma tumor cell lines

The M1152 PDX tumor cell line is a PDX tumor-derived gene from NSG mice implanted with melanoma tumor fragments. The PDX melanoma cell line was characterized by measuring melanoma tumor-specific markers called melanoma-associated chondroitin sulfate proteoglycan (MCSP) and PD-L1 expression via flow cytometry. Approximately 80% of the tumor cells expressed MCSP protein, indicating that the majority of these cells were of melanoma origin. IFN-γ treatment induced an upregulation of PD-L1 from 13% to 82% within 48 hours. This encapsulates an in vivo drug resistance mechanism in which tumor cells increase PD-L1 expression in response to T cell secretion of IFN-γ during tumor cell lysis and supports the relevance of the M1152 PDX model for assessing M1152 PD-1 KO TIL activity.

In vivo tumor burden control of M1152 PD-1KO TIL

To determine the effect of PD-1KO TILs on tumor growth, PD-1KO or TIL-mimicking cells were adoptively transferred into hIL-2NOG mice implanted with autologous melanoma cells. Combinations of anti-PD-1 and TIL-mimicking cells served as controls for PD-1/PD-L1 pathway blockade. The in vivo efficacy of PD-1KO TILs was assessed by tumor burden reduction.

Figure 72 shows the in vivo antitumor activity of the M1152 PD-1KO TIL product. hIL-2NOG mice (n=14 per treatment group) transplanted with melanoma tumor cells underwent adoptive metastasis with either PD-1KO or a TIL-mimicking agent. Anti-PD-1 antibody treatment in combination with a TIL-mimicking agent was included as a control for PD-1/PD-L1 blockade. Tumor volume was calculated as length × width and plotted as a function of time post-implantation. Statistical significance was assessed using the Wilcoxon rank-sum test, with *, **, and **** indicating p-values <0.05, <0.01, and <0.0001, respectively, and considered statistically significant.

Tumor reduction was observed in mice treated with PD-1KO and TIL-mimicking antibodies, with or without anti-PD-1 antibody therapy. This indicates that TILs can recognize and lyse transplanted tumor cells, thus validating the tumor-specific recognition of TILs via MHC and TCR conjugation. Furthermore, even in combination with anti-PD-1 therapy, PD-1KO TILs induced superior tumor control relative to TIL-mimicking TILs. The failure of anti-PD-1 to enhance the in vivo efficacy of TIL-mimicking TILs in immunodeficiency and the lack of primary and secondary lymphoid organs in hIL-2NOG mice suggests that the in vivo mechanism of systemic PD-1 suppression may require an intact immune system. Conversely, the improved antitumor activity of PD-1KO TILs compared to TIL-mimicking TILs indicates that endogenous PD-1 suppression confers a functional advantage on TILs.

Therefore, this study demonstrates that TALEN-mediated PD-1KO confers a functional advantage on TILs. This finding is consistent with several published studies that describe an enhanced ability of PD-1KO T cells to control tumor growth relative to unmodified T cells (Menger 2016, Marotte 2020).

Figures 73A-73B show the persistence of TALEN protein in autologous TILs as a function of time, as measured by Western blotting. TILs generated from melanoma (n=6; M1011, M1017, M1025, M1030, M1034, M1040) were electroporated with PD-1 TALEN mRNA. NE TILs were used as a baseline control. TALEN protein was detected in whole-cell extracts from TILs by Western blotting at 8, 10, 12, 14, and 18 hours post-electroporation (Figure 73A) and on days 1, 2, 4, and 6 (Figure 73B). The intensity of the TALEN-specific bands was quantified by densitometric analysis, with the resulting OD values averaged over time and background corrected for the mean values of NE TILs. The mean values are plotted as bars, and the standard error is shown as a vertical line.

Quantification of mutagenesis at target sites and candidate off-target sites in PD-1 TALEN

Information about each candidate off-target site is included in Table 59.

Table 59. Off-target site analysis

IL-2-independent proliferation assay

By day 28 of analysis, none of the PD-1KO TIL product samples evaluated using CD3/CD28 activation proliferated in the absence of IL-2. All samples cultured in the presence of IL-2 proliferated by day 28, with a minimum amplification of 1.7-fold and a maximum of 407-fold. Notably, paired TALEN-edited TILs and unmodified TILs isolated from the same tumor showed comparable overall amplification folds across samples. These results demonstrate the safety of the PD-1KO TIL drug product by confirming the absence of cell proliferation in the absence of IL-2, indicating no malignant transformation of cells in this product.

Cytogenetics/karyotype analysis

Two aberrant karyotypes were observed, with X chromosome monosomy detected in 4–5 of the 20 metaphase diffuses analyzed. These structural abnormalities were detected in both non-genome-edited TIL samples and TALEN-edited TIL samples, therefore, these abnormalities cannot be attributed to TALEN-mediated genome editing. Furthermore, as assessed in a cytogenetic analysis of 200 metaphase cells, no significant increase in mutagenesis was observed in PD-1 TALEN-edited TILs compared to unmodified TILs. Based on these results, it is concluded that no evidence of TALEN-induced clonal chromosomal aberrations was found in any of the tested samples, and therefore, no significant genotoxicity was identified following TALEN-mediated genome editing.

Example 21: Exemplary TIL Manufacturing Process

Figures 74A-F depict an exemplary TIL manufacturing process. Briefly, on day 0, tumor tissue is isolated from hypothermosol, the bioload sample is stored in transport medium, and tumor fragments are seeded into multiple (2, 3, or 4) G-Rex 100MCS culture flasks at a density ≤50 fragments/flask. Excess fragments are rapidly frozen. In some embodiments, the activation step can be incorporated into the pre-REP step, which provides a better co-stimulatory environment for TILs within the rosette/tumor ME. For example, on day 3, 60 μg of OKT3 or TransAct is added to each of the multiple G-Rex 100MCS culture flasks, and the cells undergo initial activation. On days 7/8, the volume is reduced, the sample is filtered, and transferred to a pooled EXP1000. The sample is removed for cell counting/viability analysis. Cells are washed, centrifuged at 400 g, 20 °C for 10 min, and aliquoted into TALEN and control samples at a ratio ≥9:1. Cells were resuspended in T buffer and electroporated at 10 × ^10⁶ cells/cup with TALEN mRNA (TALEN sample) or without RNA (control sample). Electroporation control and TALEN samples from each cup were seeded into G-Rex100M culture flasks containing 100 mL of medium (containing 6000 IU/mL IL-2) and incubated at 37°C for 1 hour. Feeder cells were irradiated, thawed, collected, and incubated with IL-2. Samples were removed for cell counting and viability analysis. Feeder cells and additional IL-2 and OKT3 were added to the incubated control and TALEN samples in G-Rex 100MCS culture flasks to generate REP cultures (1x G-Rex 100MCS for control REP culture, ≤9x G-Rex 100MCS for TALEN REP culture). On days 10/11, 500 mL of culture medium along with 3000 IU/mL IL-2 was added to each culture flask of the REP culture. Alternatively, the cell culture medium could be completely replaced with fresh medium. This step did not require transferring the cell suspension and did not use G-Rex 500MCS. On day 16, the volume was reduced, and the samples were collected. The samples were transferred via a blood filter, and the samples were removed for cell counting/viability analysis. Control samples were centrifuged, and the control final formulation was prepared and cryopreserved at a controlled rate. TALEN samples were processed using the LOVO system, and the TALEN residue final formulation was prepared and cryopreserved at a controlled rate. The prepared product samples were then subjected to quality control analysis.

This process has the following advantages: a) it eliminates the suspension transfer step for activation in the bag; b) it allows for 1/10 scale control; c) it eliminates in-process transfer to G-Rex10; d) it eliminates the overnight incubation step at 30°C, allowing for immediate reactivation (REP) of the medium at ambient temperature; and e) it eliminates one treatment day.

Example 22: A Phase 1/2 Study Evaluating the Efficacy and Safety of PD-1KO TIL Infusion

introduction

The phase 1/2 open-label study of PD-1 knockout tumor-infiltrating lymphocyte (PD-1KO TIL) products was conducted in participants with unresectable or metastatic melanoma or stage III or IV non-small cell lung cancer.

PD-1KO TIL product is an autologous TIL product that has undergone genome editing with transcription activator-like effector nucleases to disrupt the gene PDCD1 encoding PD-1. This protocol will evaluate PD-1KO TIL product as a treatment option for participants with unresectable or metastatic melanoma or stage III or IV NSCLC.

Research Principles

This study marks the first human investigation of the PD-1KO TIL product. The antitumor activity of the PD-1KO TIL product and its ability to directly target and kill tumor cells in a manner similar to non-genome-edited autologous TIL products were evaluated, with the potential for enhanced antitumor activity due to PDCD1 disruption.

Consistent with the hypothesis, enhanced antitumor activity of the PD-1KO TIL product was observed relative to non-genome-edited autologous TILs in a patient-derived xenograft mouse model. A proof-of-concept nonclinical study of the PD-1KO TIL product further demonstrated that TALEN-mediated PDCD1 mutagenesis was reproducible, correlated with reduced PD-1 cell surface expression, and indicated that the general biological characteristics of autologous TILs considered crucial for antitumor activity were either unaffected or positively affected by PD-1 gene knockout. In the absence of conventional nonclinical animal models associated with human TIL testing, in vitro studies supported the safety of the PD-1KO TIL product. Low off-target activity was detected at one of the top 20 potential off-target sites. Given the low mutation frequency and the fact that this site corresponds to a non-coding RNA not known to be associated with the disease, this is unlikely to pose a safety risk to the product. Additional in vitro studies showed no TALEN-related clonal chromosomal abnormalities following TALEN-mediated genome editing.

background

Unresectable or metastatic melanoma, as well as stage III and IV NSCLC, represent malignancies with a significant unmet medical need. Despite recent advances in melanoma treatment, including ICIs (e.g., anti-cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4] and anti-PD-1 monoclonal antibodies [mAb]) and targeted therapies (e.g., proto-oncogene B-Raf [BRAF] and mitogen-activated protein kinase [MEK] inhibitors), unresectable or metastatic melanoma remains difficult to treat and continues to be a major public health problem (Howlader 2020; Steininger 2021). While several effective NSCLC therapies are currently available, including ICIs (i.e., nivolumab and pembrolizumab), platinum-based double chemotherapy, and tyrosine kinase inhibitors (TKIs) or other targeted therapies against driver mutation-positive tumors, none have provided adequate activity and durable cancer control. Ultimately, most tumors progress despite the initial effectiveness of the available therapies. Therefore, despite the approval of ICI, there remains a significant unmet medical need for melanoma and NSCLC patients who have made progress on available standard care therapies.

Autologous TILs have demonstrated durable responses and have the potential to address a major unmet need in clinical trial participants with metastatic melanoma who have limited treatment options, including those with primary refractory to anti-PD-1/PD-L1 therapy (Rosenberg 2011; Larkin 2021; Sarnaik 2021). Similarly, preliminary data suggest that TILs elicit clinically meaningful responses in NSCLC participants who have experienced disease progression following a series of therapies, including ICIs, and in participants with epidermal growth factor receptor (EGFR) driver mutation-positive tumors (Creelan 2021; Schoenfeld 2021).

The antitumor activity of TILs can be inhibited through interactions between lymphocytes and the tumor microenvironment. Specifically, signaling that occurs when PD-1 on the surface of T cells binds to PD-L1 on tumor cells negatively impacts T cell antitumor activity. Consistent with this finding, various antibody therapies that specifically block the PD-1/PD-L1 pathway, such as the anti-PD-1 antibodies pembrolizumab and nivolumab, have shown potent activity in solid tumors, both as single agents and in combination with non-genome-edited autologous TILs. Notably, in addition to their promising anticancer activity, clinical studies of non-genome-edited autologous TIL therapy combined with anti-PD-1 antibodies in unresectable or metastatic melanoma, recurrent and/or metastatic head and neck squamous cell carcinoma, and persistent, recurrent, or metastatic cervical cancer provide preliminary evidence of the safety of PD-1 signaling inactivation in the context of adoptive cell therapy (ACT) with TILs, suggesting a safety profile consistent with that of individual therapies (O'Malley 2021). This is consistent with the results of other studies conducted to date in participants with metastatic melanoma (evaluating the combination of TIL therapy and immune checkpoint inhibition) (Besser 2013; Mullinax 2018) and a study investigating the administration of TIL therapy in combination with nivolumab in participants with NSCLC (Creelan 2021).

Systemic administration of anti-PD-1 antibodies generates adverse events (AEs) due to the non-specific upregulation of immune pathways via T cell activation and proliferation. As a potential alternative to this treatment, TALEN genome editing technology is used to specifically inactivate PDCD1 in autologous TILs, thereby producing PD-1KO TIL products, PD-1 gene knockout products. By limiting the inhibition of PD-1 signaling in therapeutic tumor-derived T cells, PD-1KO TIL product therapy is expected to provide a safer alternative to ICI therapy, which imparts systemic inhibition of the PD-1 pathway. Furthermore, compared to anti-PD-1 antibody regimens requiring repeated administration, PD-1KO TIL product therapy has the potential to offer clinical benefit as a one-time treatment.

Benefit/Risk Assessment

Currently, no clinical safety data are available for the PD-1KO TIL product, as this is the first human study of the PD-1KO TIL product. There are no clinical safety data from studies of ACT and autologous TILs with PD-1 gene knockout via genome editing; however, safety data are available from non-genome-edited TIL regimens administered alone or in combination with anti-PD-1 antibodies. Safety data from studies of non-genome-edited TIL products are considered relevant because they are manufactured using a derivative process of the PD-1KO TIL product manufacturing process, have the same formulation as the PD-1KO TIL product, and are administered as part of a regimen similar to the proposed PD-1KO TIL product regimen.

The risk and benefit assessment presented below is based on observations related to the administration of NMA-LD and IL-2, as reported in their respective prescribing information, and clinical experience with similar treatments to PD-1KO TIL products, specifically non-genome-edited autologous TIL products administered alone or in combination with anti-PD-1 antibodies, as part of a regimen similar to the proposed PD-1KO TIL product regimen.

Benefit assessment

Potential benefits to participants include the possibility of receiving a research intervention that could stop, slow, or reverse cancer progression in patient populations lacking effective treatment options.

Overall Benefits and Risks Conclusion

Considering the following, the benefit-risk profile of the PD-1KO TIL product treatment regimen is suitable for participants planned to be enrolled in the IOV-GM1-201 study:

●The known toxicities of non-genome-edited autologous TIL regimens (i.e., NMA-LD, IL-2, and non-genome-edited autologous TIL products) administered alone or in combination with anti-PD-1 antibodies are monitorable and manageable.

● Measures taken to minimize the risk to participants

● The lack of effective treatment options that provide durable benefits to the study population, and

●This new therapy has the potential to improve the antitumor activity and safety profile of existing TIL-based solid tumor treatment regimens and PD-1 inhibitor therapies.

Goals and End Points

Research Design

Overall Design

This is a phase 1/2, open-label, non-randomized, multi-cohort, multicenter study with a safety lead-in period, evaluating autologous TIL regimens using the PD-1KO TIL product in participants with unresectable or metastatic melanoma (cohort 1) or stage III or IV NSCLC (cohort 2). In each cohort, tumor samples were excised from each participant and cultured in vitro to produce IOV-4001. Participants were infused with the PD-1KO TIL product, followed by IL-2, after lymphocyte depletion chemotherapy including cyclophosphamide and fludarabine. During the safety lead-in period, the first three participants from either cohort will receive treatment in a staggered manner. Once each participant successfully completes the 28-day DLT observation period (starting at the time of PD-1KO TIL product infusion), the next participant may be authorized to continue with NMA-LD. Subsequent recruitment will follow a standard 3+3 downgrade design, allowing additional participants to join while evaluating the safety and tolerability data of the emerging PD-1KO TIL product. These safety data will be reviewed by the SRC, which comprises representatives of the sponsor and researchers from all recruited DLT-evaluable participants, to advise on dose level decisions. Overall safety will be monitored by an independent DSMB. Recruitment for both cohorts will continue after the first 3 to 6 participants have completed the DLT observation period and received DSMB approval; there will be no staggered periods between participants.

Scientific Principles of Research Design

The study was designed without a control group because these participants, who had undergone extensive pretreatment, had no alternative effective treatment options that could provide durable benefits to the study population. Although no comparisons were available, given the nature of the participants' advanced, refractory disease and the fact that the study intervention (autologous TIL regimen) was administered as a single, one-time therapy, any major tumor regression observed during the study could be presumed to be attributable to the study intervention.

The primary efficacy endpoint of the second phase of this study, namely the ORR according to RECIST v1.1, is the validated method for evaluating tumor response in a single-arm study.

Study population

Prospective approval of deviations from recruitment criteria is not permitted; this is also known as program waiver or exemption.

Key inclusion criteria

Participants are eligible to be included in the study only if they meet all of the following criteria:

1. Participants must be at least 18 years old when signing the informed consent form.

2. Participants who were histologically or pathologically diagnosed with unresectable or metastatic melanoma of stage IIIC, IIID, or IV (cohort 1) or stage III or IV NSCLC (cohort 2).

3. Participants who have previously received the following treatments:

Cohort 1 (Melanoma): Participants who experienced documented radiographic disease progression during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibody. If the tumor was BRAF V600 mutation-positive, participants also received a BRAF inhibitor with or without a MEK inhibitor.

Cohort 2 (NSCLC): Participants who had previously received no more than 3 lines of prior therapy, and

● Participants without oncogene-driven tumors who experienced documented progression of radiation-related disease during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibodies.

● Or for participants with tumors driven by oncogenes for which effective targeted therapies are available (e.g., EGFR, anaplastic lymphoma kinase [ALK], ROS proto-oncogene [ROS], or proto-oncogenes encoding MET tyrosine receptor kinase [MET]), who have experienced documented radiological disease progression during or after at least one line of appropriate targeted therapy and platinum-based dual chemotherapy, or during systemic therapy with PD-1/PD-L1 blocking antibodies, or within 12 weeks after the last dose of a PD-1/PD-L1 blocking antibody.

4. Participants whose ECOG fitness level is 0 or 1.

5. Participants assessed as having at least one resectable lesion

6. Previously irradiated lesions must have radiographic progression before collection.

7. Participants who are of childbearing age or whose partners are of childbearing age must be willing to use an approved, highly effective method of contraception during the treatment period and for 12 months after receiving all protocol-related treatments.

Key Exclusion Criteria

Participants were excluded from the study if they met any of the following criteria:

1. Participants with severe heart disease.

2. Participants with uveal/ocular melanoma.

3. Participants with symptomatic and untreated brain metastases.

4. Participants with any form of primary immunodeficiency.

5. Participants who have had another primary malignant disease within the previous 3 years.

6. Participants who have received or will receive a live or attenuated vaccine within 28 days prior to the start of NMA-LD.

References for Example 22

Besser MJ, Shapira-Frommer R, ltzhaki O, et al. Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma: Intent-to-Treat Analysis and Efficacy after Failure to Prior Immunotherapies. ClinCancer Res. 2013; 19(17): 4792-800.

Creelan BC, Wang C, Teer J, et al. Tumor-Infiltrating LymphocyteTreatment for Anti-PD-1-Resistant Metastatic Lung Cancer: A Phase 1 Trial. NatMed. 2021; 27(8): 1410-1418.

Howlader N, Noone A, Krapcho M, et al. 2020. SEER Cancer Statistics Review, 1975-2017. National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2017/, based on November 2019 SEER data submission.

Larkin JMG, Sarnaik A, Chesney JA, et al. Lifileucel(LN-144), aCryopreserved Autologous Tumor Infiltrating Lymphocyte(TIL)Ther apy inPatients with Advanced Melanoma: Evaluation of Impact of Prior Anti-PD-1Therapy.J Clin Oncol.2021;39(15_suppl):9505.

Mullinax JE, Hall M, Prabhakaran S, et al. Combination of lpilimumab and Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes for Patients withMetastatic Melanoma. Front Oncol. 2018; 8:44.

O'Malley D, Lee SM, Psyrri A, et al.Phase 2Efficacy and Safety ofAutologous Tumor-Infiltrating Lymphocyte(TIL)Cell Therapy in Combinati on with Pembrolizumab in Immune Checkpoint Inhibitor-Patients with Advanced Cancers J Immunother Cancer. 2021; 9(suppl 2, abs492): A523-4.

Rosenberg SA, Yang JC, Sherry RM, et al. Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell TransferImmunotherapy. Clin Cancer Res. 2011;17(13):4550-7.

Sarnaik AA, Hamid O, Khushalani NI, et al. Lifileucel, a Tumor-Infiltrating Lymphocyte Therapy, in Metastatic Melanoma. J Clin Oncol. 2021: JCO2100612.

Schoenfeld AJ, Lee SM, Paz-Ares L, et al. First Phase 2Results of Autologous Tumor-Infiltrating Lymphocyte (TIL; LN-145) Monotherapy in Patient swith Advanced, Immune Checkpoint Inhibitor-Treated, Non-Small Cell Lung Cancer (NsCLC). J Immunother Cancer. 2021; 9 (suppl2, abs 458): A486-7.

Steininger J, Gellrich FF, Schulz A, et al. Systemic Therapy of Metastatic Melanoma: On the Road to 75Cure. Cancers (Basel). 2021; 13(6).

Example 23: Evaluation of Xenon Electroporation Instrument

A study was conducted to determine whether a thermoexcimer could be used in the process of generating PD-1KO TILs. The results are summarized in Figure 76.

Method Overview

Activated or pre-REP TILs are used for experiments.

●REP TIL: Demonstration Day, Neon Experiment 1, Xenon Experiment 1

●Pre-REP TIL: Xenon Experiment 3, Xenon Experiment 4

●Activate for 5 days using TransAct

Cell preparation days for electroporation (same across experiments):

● Equilibrate the buffer solution to room temperature.

● Wash TIL with 1x PBS

● Wash TIL with 1x electroporation buffer (Thermo-CTS ^™ Xenon ^™ Genome Editing Buffer)

● Centrifuge and resuspend in the final volume of electroporation buffer.

○ A single injection requires an exact volume of 1 ml per sample (maximum 100e6/ml).

○ The neon volume can be 10ul or 100ul (maximum 20e6/ml).

●Add mRNA (GFP 1ug/1e6 cells, TALEN 4ug/1e6 cells)

●Electroplution

● Rest overnight in CM2 + 3000 IU/ml IL-2 in G-Rex10 (if using >10e6, split into 2 G-Rex10s).

●37℃, for GFP mRNA

●30℃, for TALEN mRNA

● Collect resting samples and bacterial strains using a 150µm cell filter.

● Perform cell counting and flow staining or REP (Xenon Experiment 4).

Experiment 1: Demonstration Day, Thermo

● Activated REP TIL (L4346) was tested using GFP mRNA.

● Thawing (Day 0) and Activation (Day 1) 50e6+TransAct

● 1ug/1e GFP mRNA per 6 cells (stock solution, 1mg/ml)

● Electroporation using Thermo's recommended optimization scheme

● Single injection (range: 2e7-1e8 cells per 1ml) – 20e6/ml

●Neon – 2e6/100ul

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 77, the results indicate that after 24 hours, 1700/20/1 and 2300/3/4 showed higher GFP expression and recovery rates.

Experiment 2: Neon Experiment 1

●Objective: To optimize two electroporation setups identified during the xenon demonstration using neon and compare them with BTX.

●Cell concentration: 2e6 cells per 100 μl of neon-1

● Xenon backbone electroporation setup:

○1700/20/1

○2300/3/4

● Change the duration (ms) and number of pulses while keeping the voltage setting constant.

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 78, the results indicate that the 2300/3/4 Thermo recommended setting (5*) has the highest GFP expression and 60% recovery rate, and the recovery rate is improved at the 2300V voltage setting with shorter duration (2ms) and fewer pulses (3), and GFP expression is reduced.

Experiment 3: Xenon Experiment 1

●Objective: To determine the effect of cell concentration on xenon-GFP expression and recovery rate.

●N = 1 activated REP TIL sample (EP11231)

●Xenon and BTX

●Electroporation conditions: 2300/3/4, selected based on maximum GFP expression and 60% recovery rate.

●Use a single injection to test cell concentration

○1e6/ml

○5e6/ml

○10e6/ml

○25e6/ml

●BTX Comparison

○10e6+GFP (in 400 μl, therefore the concentration is 25e6/ml)

○2e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 79, the results indicate that GFP expression was slightly reduced when xenon was used compared to BTX. GFP expression was not affected by cell concentration, but the recovery rate decreased and apoptosis increased at lower cell concentrations. After 24 hours, 2300/3/4 appeared to be toxic at low cell concentrations.

Experiment 4: Xenon Experiment 3

Objective: To determine the effect of cell concentration (low/high) on xenon GFP expression and recovery rate using the EP setting that exhibits the highest recovery rate.

●N = 1 activated pre-REP TIL sample (K7136)

●Xenon and BTX

●Electroplution conditions:

○1400/30/1

○1700/20/1

○2300/2/3

○2500/2/5

●Use a single injection to test cell concentration

○5e6/ml

○25e6/ml

●BTX Comparison

○5e6+GFP (in 200 μl, therefore the concentration is 25e6/ml)

○1e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 80, the results indicate that high GFP expression exists between samples, comparable to that of BTX, and the difference between 5e6 and 25e6 is minimal. For EP settings 1400/30/1 and 2300/2/3, there are similar recovery rates between 5e6 and 25e6, and after 24 hours, the recovery rate of 2300/2/3 is comparable to that of BTX.

Experiment 5: Xenon Experiment 4

●Objective: To compare the Xenon electroporator and the BTX electroporator using TALEN mRNA.

●N = 1 activated pre-REP TIL sample (L4374)

●Xenon and BTX

●Electroporation conditions:

○2300/2/3

●Conditions:

Xenon (Talen(25e6))

Xenon simulant (5e6)

○BTX Talen (25e6)

○BTX emulator (5e6)

●Test parameters:

○Resuscitation rate and survival rate

○ PD-1 KO based on flow cytometry

■ PD-1 phase KO flow cytometry data operated on resting samples after electroporation (without REP)

■REP collects final PD-1KO flow cytometry data from samples

As shown in Figure 81, the results showed that the recovery rates of xenon and BTX were similar between 5e6 and 25e6, with xenon having a slightly higher recovery rate. The PD-1 KO efficiency (resting cells after electroporation) was similar between xenon (89%) and BTX (86%). Furthermore, for the analysis of PD-1 expression and KO efficiency in REP samples, the KO efficiency of xenon (83%) was slightly higher than that of BTX (76%).

in conclusion

The study identified an electroporation setting of 2300/2/3 for xenon that resulted in high cell resuscitation rates (comparable to BTX) at both low and high cell concentrations, high GFP expression (comparable to BTX) at both low and high cell concentrations, and high PD-1 KO efficiency (comparable to BTX).

The above embodiments are provided to give those skilled in the art a complete disclosure and description of how to prepare and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications to the above-described patterns of the invention that are obvious to those skilled in the art are intended to fall within the scope of the following claims. All patents and disclosures mentioned in this specification indicate the skill level of those skilled in the art to which this invention pertains.

All headings and section titles are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different headings and sections as needed, in accordance with the spirit and scope of the invention described herein.

All references cited in this article are incorporated herein by reference in their entirety for all purposes, as if each individual publication or patent or patent application were specifically and individually indicated to be incorporated herein by reference in their entirety for all purposes.

As will be apparent to those skilled in the art, various modifications and alterations can be made thereto without departing from the spirit and scope of this application. The specific embodiments and examples described herein are provided by way of example only, and this application is limited only to the appended claims and the full scope of their equivalents.

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 63/242,373, filed September 9, 2021; U.S. Provisional Application No. 63/287,670, filed September 9, 2021; U.S. Provisional Application No. 63/322,190, filed March 21, 2022; U.S. Provisional Application No. 63/354,605, filed June 22, 2022; and U.S. Provisional Application No. 63/394,248, filed August 1, 2022, the disclosures of which are incorporated herein by reference in their entirety.

Background of the Invention

Using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) to treat large, refractory cancers represents an effective approach for treating patients with poor prognoses. (Gattinoni et al., Nat. Rev. Immunol. 2006, 6, 383-393). TILs are predominantly T-cells, and IL-2-based TIL expansion followed by a “rapid expansion process” (REP) is the preferred method for TIL expansion due to its speed and efficiency. (Dudley et al., Science 2002, 298, 850-54; Dudley et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. Immunother. 2003, 26, 332-42.) Numerous approaches have been explored to improve the response of melanoma to TIL therapy and to extend TIL therapy to other tumor types, but success has been limited and the field remains challenging. (Goff et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg et al., Clin. Cancer Res. 2011, 17, 4550-57.) Combination studies with single immune checkpoint inhibitors have also been described, but further investigation is underway and additional treatment options are needed (Kverneland et al., Oncotarget, 2020, 11(22), 2092-2105).

Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, aseptic issues, and other factors described herein, severely restricting their therapeutic potential for patients refractory to checkpoint inhibitor therapy. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes, suitable for treating patients with few or no viable treatment options. This invention addresses this need by providing a shortened manufacturing process for generating TILs.

The present invention provides improved and/or shortened processes and methods for amplifying TILs and generating therapeutic TIL populations, including methods for gene editing at least a portion of the therapeutic TIL population to enhance its therapeutic efficacy.

Summary of the Invention

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing of at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, the culturing or rapid second expansion of the fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the fourth TIL population into multiple subcultures; each subculture being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs or a therapeutic TIL population.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the activation of the second TIL population is performed using anti-CD3 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using OKT-3.

In some implementations, the step of activating the second TIL population is performed using 300 ng/mL of OKT-3.

In some implementations, the step of activating the second TIL population is performed using anti-CD3 agonist and anti-CD28 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using TransAct.

In some implementations, the step of activating the second TIL population is performed using TransAct diluted at 1:10, 1:17.5, or 1:100.

In some implementations, the step of activating the second TIL population takes about 2 days.

In some implementations, the step of activating the second TIL population takes about 3 days.

In some implementations, the step of activating the second TIL population takes approximately 4 days.

In some implementations, the step of activating the second TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the fourth TIL population takes about 8 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 9 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the step of culturing the fourth TIL population takes about 10 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A second TIL population was produced by culturing a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium for about 3–9 days in a first cell culture medium containing IL-2 and OKT-3;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some implementations, the step of culturing or rapidly expanding the third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the third TIL population into multiple subcultures; each of the subcultures being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the third TIL population takes about 8 days.

In some implementations, the step of culturing the third TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes about 8-9 days.

In some implementations, the step of culturing the third TIL population takes about 10 days.

In some implementations, the step of culturing the third TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, the step of culturing or initially amplifying the first TIL population in a first culture medium also includes anti-CD3 and anti-CD28 beads or antibodies.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population for about 3 days in a first cell culture medium containing IL-2 and anti-CD3 and anti-CD28 beads or antibodies, followed by culturing the first TIL population for 2-4 days in a cell culture medium containing IL-2 and OKT-3.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of at least two gene editors is transferred independently of the transfer of any other gene editor during the electroporation step.

In some implementations, the electroporation step also includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing IL-2 and/or IL-15.

In some embodiments, the first and second resting periods comprise incubating a third or fourth TIL population in a second cell culture medium containing 300 IU/mL, 1000 IU/mL, or 6000 IU/mL of IL-2.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing 15 ng/mL IL-15.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first and second resting periods comprise incubating a third or fourth TIL population at approximately 25, 28, 30, 32, 35, or 37°C with approximately 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some implementations, the electroporation step is preceded by washing a second or third TIL population in a cell pore buffer.

In some embodiments, the at least one gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates PD-1 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CTLA-4 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates LAG-3 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CISH expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CBL-B expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CTLA-4.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and TIGIT.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CISH and CBL-B.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, the tumor tissue is processed into multiple tumor fragments.

In some implementations, the tumor fragment is added to the closed system.

In some embodiments, 150 or fewer fragments, 100 or fewer fragments, or 50 or fewer fragments are added to the closed system. 167. A gene-edited tumor-infiltrating lymphocyte (TIL) population comprising an expanded TIL population, wherein the expression of at least one protein is regulated by a gene editor transferred to at least a portion of the expanded TIL population.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one protein is PD-1.

In some embodiments, the at least one protein is CTLA-4.

In some embodiments, the at least one protein is LAG-3.

In some embodiments, the at least one protein is CISH.

In some embodiments, the at least one protein is CBL-B.

In some embodiments, the at least one protein is TIGIT.

In some embodiments, the expression of the at least two proteins is regulated by at least two gene editors transferred to at least a portion of the amplified TIL population, wherein the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of the first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of the second protein.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first TALE protein and the second TALE protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some implementations, the gene-edited TIL populations disclosed herein are created using the methods disclosed herein.

In some embodiments, this document provides a pharmaceutical composition comprising the gene-edited TIL population disclosed herein and a pharmaceutically acceptable vector.

In some implementations, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of the gene-edited TIL population disclosed herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) The tumor fragment is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step includes delivering a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of the TALEN system for inhibiting CTLA-4 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CBL-B and TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is about 1× ^10⁹ to about 1× ^10¹¹ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of the collected TIL population in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the patient has been given a therapeutically effective dose of the collected TIL population in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Attached Figure Description

Figure 1: Exemplary Gen 2 (Process 2A) diagram, which provides an overview of steps A through F.

Figures 2A to 2C: Process flow diagrams of one implementation of Gen 2 (process 2A) for TIL manufacturing.

Figure 3: A diagram showing one embodiment of an exemplary manufacturing process for cryopreserved TILs (approximately 22 days).

Figure 4: A diagram showing one implementation of Gen 2 (Process 2A, a 22-day process for TIL manufacturing).

Figure 5: Comparison table of steps A to F of an exemplary implementation of process 1C and Gen 2 (process 2A) for TIL manufacturing.

Figure 6: A detailed comparison of one implementation of Process 1C and one implementation of Gen 2 (Process 2A) for TIL manufacturing.

Figure 7: Exemplary Gen 3 type TIL manufacturing process.

Figures 8A through 8P: A) A comparison between an implementation of the 2A process (approximately 22 days) and a Gen 3 process (approximately 14 to 16 days) for TIL manufacturing. B) An exemplary Gen 3 process diagram providing an overview of steps A through F (approximately 14 to 16 days). C) Diagrams of three exemplary Gen 3 processes, outlining steps A through F (approximately 14 to 16 days) for each of the three process variations. D) An exemplary modified Gen 2-like process providing an overview of steps A through F (approximately 22 days). E-P) Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 9: Experimental flowchart providing comparability between Gen 2 (process 2A) and Gen 3 processes.

Figure 10: A comparison of various Gen 2 (Process 2A) and Gen 3.1 process implementation schemes.

Figure 11: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 12: An overview of the culture medium conditions for one implementation of the Gen 3 process (referred to as Gen 3.1).

Figure 13: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 14: A table comparing various features of the implementation schemes for the Gen 2 and Gen 3.0 processes.

Figure 15: A table showing the culture medium used in various implementation schemes of the described amplification process.

Figure 16: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 17: A schematic diagram of an exemplary implementation of a method for amplifying T cells from hematopoietic malignancies using the Gen 3 amplification platform.

Figure 18: Structures IA and IB are provided. The cylinder refers to an individual polypeptide-binding domain. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from, for example, 4-1BBL or antibodies binding to 4-1BB, which fold to form a trivalent protein. This trivalent protein is then linked to a second trivalent protein via an IgG1-Fc (including CH3 and CH2 domains), which in turn links the two trivalent proteins together via disulfide bonds (small elongated ellipses). This stabilizes the structure and provides an agonist capable of aggregating the intracellular signaling domains of six receptors with signaling proteins to form a signaling complex. The TNFRSF-binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys sequences.

Figure 19: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 20: Process overview of an exemplary implementation of the Gen 3.1 process (16-day process).

Figure 21: A schematic diagram of an exemplary implementation of the Gen 3.1 testing process (16-day to 17-day process).

Figure 22: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 23: Comparison table of exemplary Gen 2 and exemplary Gen 3 processes.

Figure 24: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day to 17-day process) preparation timeline.

Figure 25: A schematic diagram of an exemplary implementation of the Gen 3 process (14-day to 16-day process).

Figures 26A to 26B: Schematic diagrams of an exemplary implementation of the Gen 3 process (16-day process).

Figure 27: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 28: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 29: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 30: Components of the Gen 3 implementation scheme.

Figure 31: Comparison of Gen 3 implementation flowcharts (Gen 3.0, Gen 3.1 comparison, Gen 3.1 test).

Figure 32: Shows components of an exemplary implementation of the Gen 3 process (16- to 17-day process).

Figure 33: Acceptance Criteria Table.

Figure 34: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figure 35: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figures 36A to 36D: Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 37: A schematic diagram of an exemplary implementation of the process described in Example 12.

Figures 38A-38B: In vivo efficacy of PDCD-1KO TIL. A) PDCD-1KO efficiency as assessed by flow cytometry. B) hIL-2NOG mice with melanoma tumor cells transplanted via adoptive transfer using PDCD-1KO or TIL-mimicking mice (n=14 per treatment group). Anti-PD-1 antibody treatment combined with TIL-mimicking mice served as a control for PD-1/PD-L1 blockade. Statistical significance is indicated by *p<0.05, **p<0.01, and ****p<0.0001.

Figures 39A to 39E: TIL product analysis. A) Live cell dose, B) Purity, C) Identity, D) Potency, and E) PDCD-1KO efficiency of TIL products.

Figures 40A to 40B: TIL product analysis. A) TIL differentiation and B) TIL memory.

Figures 41A to 41B: Expression of activation and inhibition of related biomarkers on PDCD-1KO TIL.

Figures 42A to 42B: IL-2-independent proliferation assay of PDCD-1KO TIL products.

Figure 43: Summary of karyotype analysis results of PDCD-1KO TIL products.

Figures 44A to 44B: Cell viability (Figure 44A) and recovery fold (Figure 44B) before electroporation.

Figures 45A to 45B: Cell recovery fold (Figure 45A) and cell viability (Figure 45B) after electroporation.

Figures 46A to 46C: Gene knockout efficiency of CD3+ (Figure 46A), CD8+ (Figure 46B), and CD4+ (Figure 46C) cells.

Figures 47A to 47B: Cell recovery fold (Figure 47A) and cell viability (Figure 47B) after electroporation.

Figures 48A to 48B: Cell recovery fold (Figure 48A) and cell viability (Figure 48B) after electroporation when using 6000 IU/mL IL-2.

Figures 49A to 49B: Cell recovery fold (Figure 49A) and cell viability (Figure 49B) after electroporation under various conditions.

Figures 50A to 50C: Gene knockout efficiency of CD3+ (Figure 50A), CD8+ (Figure 50B), and CD4+ (Figure 50C) cells.

Figure 51: Cell viability before electroporation.

Figure 52: Cell resuscitation fold before electroporation.

Figures 53A to 53B: Cell recovery fold (Figure 53A) and cell viability (Figure 53B) after electroporation.

Figures 54A to 54C: Gene knockout efficiency of CD3+ (Figure 54A), CD8+ (Figure 54B), and CD4+ (Figure 54C) cells.

Figures 55A to 55B: Cell number (Figure 55A) and survival rate (Figure 55B) after various washing steps.

Figures 56A to 56B: Cell numbers after various rotation conditions using PBS washing (Figure 56A) or Cyto washing (Figure 56B).

Figures 57A to 57B: Cell viability after various rotation conditions using PBS washing (Figure 57A) or Cyto washing (Figure 57B).

Figures 58A to 58B: Total rotation comparison of cell number (Figure 58A) and total rotation comparison of cell survival rate (Figure 58B) after various rotation conditions.

Figure 59: Percentage of cell loss after total rotation under various rotation conditions.

Figures 60A to 60C: Percentage of cell loss and survival rate during electroporation, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation.

Figures 61A to 61C: Gene knockout efficiency of CD3+ (Figure 61A), CD8+ (Figure 61B), and CD4+ (Figure 61C) cells.

Figures 62A to 62B: Cell viability (Figure 62A) and fold expansion (Figure 62B) of the REP collection.

Figures 63A to 63B: Percentage of cell loss after electroporation (Figure 63A) and cell viability (Figure 63B).

Figures 64A to 64C: Gene knockout efficiency of CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells.

Figures 65A to 65B: Folding of REP collection (Figure 65A) and cell viability (Figure 65B).

Figures 66A to 66C: Cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C).

Figure 67: Percentage of growth after 3 days of rest.

Figures 68A to 68C: PD-1 gene knockout efficiency.

Figure 69: PDCD1 gene modification according to NGS.

Figures 70A to 70B: Distribution of the TCR Vβ isotype in the CD3+PD-1- subset in the main PD-1KO TIL product and NE TIL.

Figures 71A to 71B: PD-1KO TIL effect function as measured by MLR (Figure 71A) and multifunctionality (Figure 71B).

Figure 72: In vivo antitumor activity of M1152 PD-1KO TIL product.

Figures 73A to 73B: Durability of TALEN protein in autologous TILs as a function of time, measured by Western blot.

Figures 74A-F: Exemplary TIL manufacturing process.

Figures 75A-B: Schematic diagrams of the 1/2 phase study described in Example 22.

Figure 76: Summary of data described in Example 23.

Figures 77A-D: Results of the demonstration day experiment from Example 23.

Figures 78A-C: Results of neon experiment 1 from Example 23.

Figures 79A-C: Results of Xenon Experiment 1 from Example 23.

Figures 80A-B: Results of Xenon Experiment 3 from Example 23.

Figures 81A-C: Results of Xenon Experiment 4 from Example 23.

A brief explanation of sequence listings

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of moromumab.

SEQ ID NO:3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO:4 is the amino acid sequence of adefovir.

SEQ ID NO:5 is in IL-2 form.

SEQ ID NO:6 is the amino acid sequence of Nevo interleukin-α.

SEQ ID NO:7 is in IL-2 form.

SEQ ID NO:8 is a mucin domain polypeptide.

SEQ ID NO:9 is the amino acid sequence of recombinant human IL-4 protein.

SEQ ID NO:10 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO:11 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO:12 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO:13 is an IL-2 sequence.

SEQ ID NO:14 is the sequence of the IL-2 mutant protein.

SEQ ID NO:15 is the sequence of the IL-2 mutant protein.

SEQ ID NO:16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

SEQ ID NO:17 is HCDR2 of IgG.IL2R67A.H1.

SEQ ID NO:18 is HCDR3 of IgG.IL2R67A.H1.

SEQ ID NO:19 is HCDR1_IL-2kabat of IgG.IL2R67A.H1.

SEQ ID NO:20 is HCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:21 is HCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:22 is HCDR1_IL-2clothia of IgG.IL2R67A.H1.

SEQ ID NO:23 is HCDR2 clothia of IgG.IL2R67A.H1.

SEQ ID NO:24 is HCDR3 clothia of IgG.IL2R67A.H1.

SEQ ID NO:25 is HCDR1_IL-2IMGT of IgG.IL2R67A.H1.

SEQ ID NO:26 is HCDR2 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:27 is HCDR3 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:28 is the V _H chain of IgG.IL2R67A.H1.

SEQ ID NO:29 is the heavy chain of IgG.IL2R67A.H1.

SEQ ID NO:30 is the LCDR1 kabat of IgG.IL2R67A.H1.

SEQ ID NO:31 is the LCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:32 is the LCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:33 is the LCDR1 chothia of IgG.IL2R67A.H1.

SEQ ID NO:34 is the LCDR2 chothia of IgG.IL2R67A.H1.

SEQ ID NO:35 is the LCDR3 chothia of IgG.IL2R67A.H1.

SEQ ID NO:36 is the V _L chain.

SEQ ID NO:37 is a light chain.

SEQ ID NO:38 is a light chain.

SEQ ID NO:39 is a light chain.

SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

SEQ ID NO:41 is the amino acid sequence of mouse 4-1BB.

SEQ ID NO:42 is the heavy chain of utomilumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:43 is the light chain of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:44 is the heavy chain variable region (V _H ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:45 is the light chain variable region (V _L ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:46 is the heavy chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:47 is the heavy chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:48 is the heavy chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:49 is the light chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:50 is the light chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:51 is the light chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:52 is the heavy chain of urelumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:53 is the light chain of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:54 is the heavy chain variable region (VH) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:55 is the light chain variable region (VL) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:56 is the heavy chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:57 is the heavy chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:58 is the heavy chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:59 is the light chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:60 is the light chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:61 is the light chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:62 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:63 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:64 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:65 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:66 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:67 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:68 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:69 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:70 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:71 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:72 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:73 represents the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:74 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:75 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:76 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:77 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

SEQ ID NO:78 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO:79 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 1.

SEQ ID NO:80 is the light chain variable region (V _L ) of type 1 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:81 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 2.

SEQ ID NO:82 is the light chain variable region (V _L ) of type 2 of 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:83 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:84 is the light chain variable region (V _L ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:85 is the amino acid sequence of human OX40.

SEQ ID NO:86 is the amino acid sequence of mouse OX40.

SEQ ID NO:87 is the heavy chain of the OX40 agonist monoclonal antibody taboliziumab (MEDI-0562).

SEQ ID NO:88 is the light chain of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:89 is the heavy chain variable region (V _H ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:90 is the light chain variable region (V _L ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:91 is the heavy chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:92 is the heavy chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:93 is the heavy chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:94 is the light chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:95 is the light chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:96 is the light chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:99 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:100 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:107 is the heavy chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:108 is the light chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:109 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:110 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:117 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:118 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:119 is the heavy chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:120 is the heavy chain CDR2 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:122 is the light chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:125 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:126 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:130 is the light chain CDR1 of Hu106-222, an OX40 agonist monoclonal antibody.

SEQ ID NO:131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:133 is the amino acid sequence of the OX40 ligand (OX40L).

SEQ ID NO:134 is the soluble portion of the OX40L polypeptide.

SEQ ID NO:135 is an alternative soluble fraction of the OX40L polypeptide.

SEQ ID NO:136 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:137 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:138 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:139 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:140 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 021.

SEQ ID NO:141 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 021.

SEQ ID NO:142 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:143 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:144 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:145 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:146 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:147 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:148 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:149 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:150 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:151 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:152 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:153 is the heavy chain variable region ( _VH ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:154 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:155 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:156 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:157 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:160 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:161 is the amino acid sequence of the light chain variable region ( _VL ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:170 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:171 is the amino acid sequence of the light chain variable region (V _L ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:180 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:181 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:190 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:191 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:200 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:201 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:210 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:211 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:220 is the amino acid sequence of the heavy chain variable region (V _H ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:221 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4 inhibitor zeflimab.

SEQ ID NO:230 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:231 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:238 is an exemplary Clo05 l nuclease domain amino acid sequence.

SEQ ID NO:239 is an exemplary piggyBac(PB) transposase amino acid sequence.

SEQ ID NO:240 is an exemplary Sleeping Beauty transposase amino acid sequence.

SEQ ID NO:241 is an exemplary amino acid sequence of an overactive Sleeping Beauty (SB100X) transposase.

I. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and disclosures mentioned herein are incorporated herein by reference in their entirety.

As used herein, the terms "co-administration/co-administering," "administered in combination with/administering in combination with," "simultaneous," and "concurrent" encompass the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, such as multiple TILs) to a subject, such that both the active pharmaceutical ingredient and/or its metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of separate compositions and administration of a composition in which two agents are present are preferred.

The term "in vivo" refers to events that occur within the body of a subject.

The term "in vitro" refers to an event that occurs outside the subject's body. In vitro assays encompass cell-based assays using live or dead cells, and may also include cell-free assays that do not use intact cells.

The term "ex vivo" refers to an event involving the treatment or procedure performed on cells, tissues, and/or organs that have been removed from a subject's body. Appropriately, cells, tissues, and/or organs may be returned to the subject using surgical or therapeutic methods.

The term "rapid amplification" means that the number of antigen-specific TILs increases at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) within one week, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) within one week, or most preferably at least about 100-fold within one week. Several rapid amplification protocols are described herein.

In this article, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are TILs obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any expanded or proliferated population of TIL cells as discussed herein, including (but not limited to) main TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations may contain genetically modified TILs.

In this article, "cell population" (including TILs) refers to a large number of cells sharing common characteristics. Typically, the population size ranges from 1 × ^10⁶ to 1 × ^10¹⁰ cells, with different TIL populations containing varying numbers. For example, initial growth of primary TILs in the presence of IL-2 produces a main TIL population of approximately 1 × ^10⁸ cells. REP expansion is typically performed to provide a cell population of 1.5 × ^10⁹ to 1.5 × ^10¹⁰ cells for infusion.

In this document, “cryopreserved TIL” means TIL that has been processed and stored in the range of approximately -150°C to -60°C, whether primary, host, or amplified (REP TIL). General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, “cryopreserved TIL” may be distinguished from frozen tissue samples that can be used as a source of primary TIL.

In this article, “thawed cryopreserved TILs” means a population of TILs that were previously cryopreserved and then processed to be restored to room temperature or higher (including, but not limited to, cell culture temperatures or temperatures at which TILs can be administered to patients).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to reintroduce into patients with late-stage invasive solid tumors.

The term "cryopreservation media" refers to any culture medium that can be used for cryopreservation of cells. Such media may include those containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 media may be referred to by the trade name "CS10". CS10 media is a serum-free, animal-free medium containing DMSO. In some embodiments, CS10 media contains 10% DMSO.

The term "central memory T cells" refers to a subset of T cells in humans that are CD45R0+ and constitutively express CCR7 (CCR7 ^hi ) and CD62L (CD62 ^hi ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Upon TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are mainly found in the CD4 compartments of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

The term "effective memory T cells" refers to a subset of human or mammalian T cells, such as central memory T cells, which are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 ^lo ) and are heterogeneous or low in expression of CD62L (CD62L ^lo ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, upon antigen stimulation. Effector memory T cells are primarily found in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry a large amount of perforin.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, for example (but not limited to), closed G containers. Once a tumor segment is added to the closed system, the system remains closed to the external environment until the TIL is ready for administration to the patient.

As used herein, the terms “fragmenting,” “fragment,” and “fragmented” describe the process of destroying a tumor, including mechanical fragmentation methods such as crushing, slicing, splitting, and pulverizing tumor tissue, as well as any other methods used to destroy the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBL is separated from whole blood or apheresis products from a donor. In some embodiments, PBL is separated from whole blood or apheresis products from a donor by positively or negatively selecting for a T cell phenotype (e.g., CD3+CD45+ T cell phenotype).

The term "anti-CD3 antibody" refers to an antibody or a variant thereof that targets the CD3 receptor in the T-cell antigen receptor of mature T cells, such as a monoclonal antibody, and includes human, humanized, chimeric, murine, or mammalian antibodies. Anti-CD3 antibodies include OKT-3, also known as moromuzumab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof targeting the CD3 receptor in the T-cell antigen receptor of mature T cells, including human, humanized, chimeric, or murine antibodies, and including commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, San Diego, CA, USA) and moromumab or its variants, conserved amino acid substitutions, glycosylated forms, or biosimilars. The amino acid sequences of the heavy and light chains of moromumab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). Hybridomas capable of producing OKT-3 are deposited at the American Type Culture Collection and designated ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited at the European Collection of Authenticated Cell Cultures (ECACC) with catalog number 86022706.

Table 1. Amino acid sequence of moromuzumab (exemplary OKT-3 antibody)

The term "IL-2" (also referred to herein as "IL2") refers to a T-cell growth factor called interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in this invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses recombinant human IL-2, such as adefovir (PROLEUKIN, available from multiple suppliers, 22 million IU per single-use vial), as well as recombinant IL-2 forms supplied by CellGenix (CELLGRO GMP) of Portsmouth, NH, USA, or ProSpec-Tany TechnoGene Ltd. (catalog number CYT-209-b) of East Brunswick, NJ, USA, and other commercial equivalents from other suppliers. Adefovir (desalanyl-1, serine-125 human IL-2) is a non-glycosylated recombinant human IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of the adefovir suitable for use in this invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including the PEGylated IL-2 prodrug beempegaldesleukin (NKTR-214, as SEQ ID NO:4, PEGylated recombinant human IL-2 wherein an average of 6 lysine residues are substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluorene-9-yl)methoxy]carbonyl N ⁶ ), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO 2018/132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US2019/0275133 A1, the disclosure of which is incorporated herein by reference. The bepedelion interleukin (NKTR-214) and other PEGylated IL-2 molecules applicable to the present invention are described in U.S. Patent Application Publication No. US2014/0328791A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 applicable to the present invention are described in U.S. Patents Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations applicable to the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

In some embodiments, the IL-2 form suitable for use in this invention is THOR-707, available from Synthorx. The preparation and characteristics of THOR-707 and additional alternative forms of IL-2 suitable for use in this invention are described in U.S. Patent Application Publications Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for use in this invention is an interleukin-2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugate portion bound to the isolated and purified IL-2 polypeptide at amino acid positions selected from the following: K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the amino acid residues are numbered corresponding to SEQ ID NO: 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residues are mutated to cysteine. In some embodiments, the amino acid residues are mutated to lysine. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to non-natural amino acids. In some embodiments, the non-natural amino acids include N6-azidoethoxy-L-lysine (AzK), N6-propynylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, 3-methyl-phenylalanine, L-DOPA, fluorinated phenylalanine, Isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzoxy)-3-oxopropyl)amino)ethyl)selenoyl)propionic acid, 2-amino-3-(phenylselenoyl)propionic acid, or selenocysteine. In some embodiments, the IL-2 conjugate exhibits reduced affinity for the IL-2 receptor α (IL-2Rα) subunit relative to the wild-type IL-2 peptide. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% of the binding affinity to IL-2Rα. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 100, 200, 300, 500, 1000, or greater. In some embodiments, the conjugated moiety weakens or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugation portion comprises a water-soluble polymer. In some embodiments, each water-soluble polymer independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each water-soluble polymer independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises polydextrose, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the water-soluble polymers each independently comprise a polyamine. In some embodiments, the conjugated moiety comprises a protein. In some embodiments, an additional conjugated moiety comprises a protein. In some embodiments, the proteins each independently comprise albumin, transferrin, or transthyretin. In some embodiments, the proteins each independently comprise an Fc moiety. In some embodiments, the proteins each independently comprise the Fc moiety of IgG. In some embodiments, the conjugated moiety comprises a peptide. In some embodiments, an additional conjugated moiety comprises a peptide. In some embodiments, the peptides each independently comprise XTEN peptides, high-amino acid polymers rich in glycine (HAP), PAS peptides, elastin-like peptides (ELP), CTP peptides, or gelatin-like protein (GLK) polymers. In some embodiments, the isolated and purified IL-2 peptide is modified by glutamylation. In some embodiments, the conjugated moiety binds directly to the isolated and purified IL-2 peptide. In some embodiments, the conjugated moiety binds indirectly to the isolated and purified IL-2 peptide via a linker. In some embodiments, the linker comprises a homologous bifunctional linker. In some embodiments, the homodifunctional linker comprises Lomant's reagent, dithiobis(succinimide propionate) DSP, 3'3'-dithiobis(succinimide propionate sulfonyl succinimide) (DTSSP), succinimide disuccinate (DSS), succinimide disuccinate (BS), succinimide tartrate (DST), disulfonyl tartrate (sulfonyl DST), glycosylbis(succinimide succinate) ethylene ester (EGS), glutarate succinimide (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl hexamethylenediimide (DMA), dimethyl heptamethine (DMP), dimethyl octanediimide (DMS), dimethyl-3,3'-dithiobispropionide (DSP) DTBP), 1,4-bis(3'-(2'-pyridyldithio)propamido)butane (DPDPB), bis(cis-butenyldiimide)hexane (BMH), compounds containing aryl halides (DFDNB) (e.g., 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the connector comprises a heterogeneous bifunctional connector. In some embodiments, the heterogeneous bifunctional connector comprises 3-(2-pyridyldithio)propionic acid N-succinimide ester (sPDP), long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (LC-sPDP), water-soluble long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (sulfonyl-LC-sPDP), succinimide-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), or sulfosuccinimide-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate. (Sulfon-LC-sMPT), succinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sulfon-sMCC), m-cis-butenyldiimide benzoyl-N-hydroxysuccinimide (MBs), m-cis-butenyldiimide benzoyl-N-hydroxysulfosuccinimide (sulfon-MBs), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB) ) sulfosuccinimide aminobenzoate (sulfon-sIAB), succinimide-4-(p-cis-butenylimide-phenyl)butyrate (sMPB), sulfosuccinimide-4-(p-cis-butenylimide-phenyl)butyrate (sulfon-sMPB), N-(γ-cis-butenylimide-butyryloxy)succinimide (GMBs), N-(γ-cis-butenylimide-butyryloxy)sulfosuccinimide (sulfon-GMBs), 6-((iodoacetyl)amino)hexanoic acid succinimide (sIAX), 6-[6-(((iodoacetyl)amino)hexanoic acid succinimide Acetyl)amino)hexanoyl)amino]hexanoic acid succinimide ester (sIAXX), 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylic acid succinimide ester (sIAC), 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoic acid succinimide ester (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl reactive and thiohydrogen-reactive crosslinking agents, such as 4-(4-N-cis-butenyldiimide-phenyl)butyric acid hydrazide (MPBH), 4-(N-cis-butenyldiimide-methyl)cyclohexane- 1-Carboxy-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfon-NHs-AsA), sulfosuccinimide-(4-azidosalicylamido)hexanoate (sulfon-NHs-LC-AsA), sulfosuccinimide-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimide-4 -Azide benzoate (HsAB), N-hydroxysulfosuccinimide-4-azidobenzoate (sulfon-HsAB), N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfon-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimide-2-(m-azido-o-nitrobenzoamide)-ethyl-1,3' -Dithiopropionate (sAND), N-succinimide-4-(4-azidophenyl)1,3'-dithiopropionate (sADP), (4-azidophenyl)-1,3'-dithiopropionate N-sulfosuccinimide (sulfon-sADP), 4-(p-azidophenyl)butyrate sulfosuccinimide (sulfon-sAPB), 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate sulfosuccinimide (sAED), 7-azido-4-methylcoumarin-3-acetic acid sulfosuccinimide (sulfon-s... AMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleic anhydride group, optionally comprising a maleic anhydride hexanoyl (mc), succinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sulfon-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives thereof, or the like. In some embodiments, the conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, an additional conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in this invention is polyethylene glycol-modified as disclosed in U.S. Patent Application Publication US 2020/0181220 A1 and U.S. Patent Application Publication US 2020/0330601 A1. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion relative to one residue of SEQ ID NO:5. In some embodiments, the IL-2 form suitable for the present invention lacks the IL-2Rα linker but maintains normal binding to the intermediate affinity IL-2Rβ-γ signaling complex. In some embodiments, the IL-2 form suitable for the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5.

In some embodiments, the IL-2 form suitable for use in this invention is Nevor interleukin-α (also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes). Nevo interleukin-α, also known as the human interleukin-2 fragment (1-59) variant (Cys ¹²⁵ > Ser ⁵¹ ), is fused to the human interleukin-2 fragment (62-132) via a peptide linker ( ⁶⁰ GG ⁶¹ ), which is fused to the human interleukin-2 receptor α chain fragment (139-303) via a peptide linker ( ¹³³ GSGGGS ¹³⁸ ). It is produced in Chinese hamster ovary (CHO) cells and is glycosylated. The human interleukin-2 (IL-2) (75-133)-peptide [Cys ¹²⁵ (51) > Ser]-mutant (1-59) is fused to the human interleukin-2 (IL-2) (4-74)-peptide (62-132) via a _G2 peptide linker (60-61) and via GSG _3. The S-peptide linker (133-138) is fused to the human interleukin 2 receptor α chain (IL2R subunit α, IL2Rα, IL2RA) (1-165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, in glycoform α. The amino acid sequence of Nevo interleukin α is provided in SEQ ID NO:6. In some embodiments, Nevo interleukin α exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbers in SEQ ID NO:6), and glycosylation sites at the following positions: N187, N206, T212 (using the numbers in SEQ ID NO:6). The preparation and properties of Nevo interleukin-α, as well as additional alternative forms of IL-2 suitable for the present invention, are described in U.S. Patent Application Publication No. US2021/0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the IL-2 form suitable for the present invention has the amino acid sequence provided in SEQ ID NO:6 or its conserved amino acid substitutions. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner via a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity with IL-1Rα and having receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises an immunoglobulin comprising all or part of an Fc region, wherein the mucin domain polypeptide linker comprises an amino acid sequence of SEQ ID NO:8 or having at least 90% sequence identity with SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

Table 2. Amino acid sequence of interleukin.

In some embodiments, the IL-2 form suitable for use in this invention comprises an antibody cytokine transplantation protein comprising: a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some implementations, the IL-2 regimen comprises administering the antibody described in U.S. Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine transplantation protein comprises: a heavy chain variable region (VH) including complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region (VL) including LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into a CDR of the _VH or _VL , wherein the IL-2 molecule is a mutant protein, wherein the antibody cytokine transplantation protein preferentially amplifies T effector cells relative to regulatory T cells, and wherein the antibody further comprises an IgG heavy chain and an IgG light chain selected from the group consisting of: an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:38; an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:29; an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:29; and an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:38.

In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR1 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR2 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR3 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR1 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR2 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR3 of _VL , wherein the IL-2 molecule is a mutant protein.

The insertion of an IL-2 molecule can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence replaces all or part of the CDR sequence. The IL-2 molecule replacement can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. The IL-2 molecule replacement can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

In some embodiments, the IL-2 molecule is directly grafted into a CDR without a peptide linker, wherein there are no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted into a CDR with a peptide linker, wherein there are one or more additional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecule described herein is an IL-2 mutant protein. In some cases, the IL-2 mutant protein contains an R67A substitution. In some embodiments, the IL-2 mutant protein contains the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutant protein contains the amino acid sequence of Table 1 in U.S. Patent Application Publication No. US 2020/0270334A1, the disclosure of which is incorporated herein by reference.

In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine transplantation protein comprises HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine transplantation protein comprises a _VH region containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine transplantation protein includes a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a light chain containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a _VH region containing the amino acid sequence of SEQ ID NO:28, and a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:38, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain region containing the amino acid sequence of SEQ ID NO:38; and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 or variants, derivatives, or fragments thereof, or conserved amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with them, as described in U.S. Patent Application Publication No. 2020/0270334A1. In some embodiments, the antibody component of the antibody cytokine transplantation protein described herein comprises the immunoglobulin sequence, framework sequence, or CDR sequence of palizumab. In some embodiments, the serum half-life of the antibody cytokine transplantation protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to aldehyde interleukin or similar molecules). In some embodiments, the antibody cytokine transplantation protein described herein has a sequence as illustrated in Table 3.

Table 3: Sequence of exemplary palizumab antibody-IL-2 transplanted protein

The term “IL-4” (also referred to herein as “IL4”) refers to a cytokine known as interleukin-4, produced by Th2 T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and _IgG1 expression. Recombinant human IL-4 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-211) and ThermoFisher Scientific of Waltham, MA, USA (recombinant human IL-15 protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for this invention is provided in Table 2 (SEQ ID NO:9).

The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine called interleukin-7, which is available from stromal and epithelial cells as well as dendritic cells. (Fry and Mackall, Blood 2002, 99, 3892-904). IL-7 stimulates T cell development. IL-7 binds to the IL-7 receptor (a heterodimer composed of IL-7 receptor α and a common γ chain receptor), which belongs to a family of signaling pathways important for T cell development within the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-254) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for this invention is provided in Table 2 (SEQ ID NO: 10).

The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor called interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the contents of which are incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-230-b) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number 34-8159-82). The amino acid sequence of the recombinant human IL-15 suitable for use in this invention is provided in Table 2 (SEQ ID NO: 11).

The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the contents of which are incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-408-b) and Thermo Fisher Scientific of Waltham, Massachusetts (recombinant human IL-21 protein, catalog number 14-8219-80). The amino acid sequence of the recombinant human IL-21 suitable for use in this invention is provided in Table 2 (SEQ ID NO:21).

When indicated as “effective antitumor dose,” “effective tumor-suppressing dose,” or “therapeutic dose,” the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease. Generally, it can be stated that the pharmaceutical composition described herein, comprising tumor-infiltrating lymphocytes (e.g., passaged TILs or genetically modified cytotoxic lymphocytes), can be administered at doses of ^10⁴ to ^10¹¹ cells/kg body weight (e.g., ^10⁵ to ^10⁶ , ^10⁵ to ^10¹⁰ , ^10⁵ to ^10¹¹ , ^10⁶ to ^10¹⁰ , ^{10⁶ to 10¹¹ , 10⁷} to ^10¹¹ , ^10⁷ to ^10¹⁰ , 10⁸ to ^10¹¹ , ^10⁸ to ^10¹⁰ , ^10⁹ to ^10¹¹ , or ^10⁹ to ^10¹⁰ cells/kg body weight), including all integer values within the range. TIL (in some cases, including genetically modified cytotoxic lymphocytes) compositions may also be administered at these doses multiple times. TILs (including genetically engineered TILs in some cases) can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). The optimal dosage and treatment regimen for a particular patient can be readily determined by medical professionals by monitoring the patient’s disease symptoms and adjusting the treatment accordingly.

The term "hematological malignancy" or related terms refer to cancers and tumors of the hematopoietic and lymphoid tissues of mammals (including, but not limited to, tissues of the blood, bone marrow, lymph nodes, and lymphatic system). Hematological malignancies are also known as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancy" refers to hematological malignancies affecting B cells.

The term "liquid tumor" refers to an abnormal clump of cells that is fluid in nature. Liquid tumor cancers include (but are not limited to) leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs derived from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs derived from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

As used herein, the term “microenvironment” can refer to the solid or hematologic tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanotransmitters that “promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, encourage treatment resistance, and provide a niche for the vigorous growth of dominant metastases,” as described by Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression of the microenvironment.

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (on days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m²/day for 5 days (on days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours to achieve physiological tolerance.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for components of the immune system (“cytokine pool”) prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as “immunosuppressive modulation”) in a patient prior to the introduction of the TIL of the present invention.

The term "effective amount" or "therapeutic effective amount" refers to an amount of compound or combination of compounds as described herein that is sufficient to achieve the intended application, including (but not limited to) the treatment of a disease. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo) or the subject being treated and the disease condition (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. The term also applies to doses that will induce a specific response in target cells (e.g., reduced platelet adhesion and/or cell migration). Specific doses will vary depending on: the specific compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

The terms “treatment”, “treating”, “treat”, etc., refer to achieving a desired pharmacological and/or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of partial or complete cure of a disease and/or adverse effects attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development or progression; and (c) alleviating the disease, i.e., causing disease remission and/or alleviating one or more disease symptoms. “Treatment” is also intended to cover the delivery of agents to provide a pharmacological effect, even in the absence of a disease or ailment. For example, “treatment” covers the delivery of compositions that can induce or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

The terms “non-myeloablative chemotherapy,” “non-myeloablative lymphocyte depletion,” “NMALD,” “NMA LD,” “NMA-LD,” and any variations thereof are used interchangeably to indicate a chemotherapy regimen designed to deplete a patient’s lymphoid immune cells while avoiding depletion of their myeloid immune cells. Typically, a patient receives one cycle of non-myeloablative chemotherapy before receiving tumor-infiltrating lymphocytes as described herein.

When used in conjunction with parts of a nucleic acid or protein, the term "heterologous" indicates that the nucleic acid or protein contains two or more subsequences that are not found in nature to be identical to each other. For example, nucleic acids are often produced in a recombinant manner, having two or more sequences from unrelated genes arranged to create new functional nucleic acid sequences, such as a promoter from one source and a coding region from another source or coding regions from different sources. Similarly, a heterologous protein indicates a protein that contains two or more subsequences that are not found in nature to be identical to each other (e.g., fusion proteins).

In the context of two or more nucleic acids or polypeptides, the terms "sequence identity," "percent identity," and "sequence percent identity" (or their synonyms, such as "99% identical") refer to two or more sequences or subsequences that are identical or have the same specific percentage of nucleotide or amino acid residues when compared and aligned (introducing gaps where necessary) to achieve maximum correspondence and without considering any conserved amino acid substitutions as part of the sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. Suitable programs for determining the percentage of sequence identity include, for example, the BLAST package available from the U.S. Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California, USA), or MegAlign (available from DNASTAR) are other publicly available software programs that can be used for sequence alignment. Those skilled in the art can determine the appropriate parameters for optimal alignment using specific alignment software. In some embodiments, the default parameters of the alignment software are used.

As used herein, the term "variant" covers (but is not limited to) antibodies or fusion proteins that contain an amino acid sequence different from that of a reference antibody, differing in that one or more substitutions, deletions, and/or additions are present within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conserved substitutions in its amino acid sequence compared to that of the reference antibody. Conserved substitutions may involve, for example, substitutions of similar charged or uncharged amino acids. Variants retain the ability to bind specifically to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

In this document, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are cells obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any population of expanded or proliferated TIL cells as discussed herein, including (but not limited to) the main TILs and expanded TILs (“REP TILs”) as discussed herein, as well as “reREP TILs”. reREP TILs may include, for example, a second expanded TIL or a second additional expanded TIL (e.g., the TILs described in step D of Figure 8, including TILs referred to as reREP TILs).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to be reintroduced into patients after infiltrating solid tumors. TILs can be further characterized by potency; for example, if interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, then TILs can be considered potent. TILs can be considered potent if, for example, interferon (IFNγ) releases greater than approximately 50 pg/mL, greater than approximately 100 pg/mL, greater than approximately 150 pg/mL, or greater than approximately 200 pg/mL, greater than approximately 300 pg/mL, greater than approximately 400 pg/mL, greater than approximately 500 pg/mL, greater than approximately 600 pg/mL, greater than approximately 700 pg/mL, greater than approximately 800 pg/mL, greater than approximately 900 pg/mL, or greater than approximately 1000 pg/mL.

The term "deoxyribonucleic acid" encompasses both natural and synthetic, unmodified and modified deoxyribonucleic acids. Modifications include alterations to the linkages between deoxyribonucleic acids in the sugar moiety, base moiety, and/or oligonucleotides.

The term "RNA" is defined as a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" is defined as a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, RNA produced in a recombinant manner, and altered RNA that differs from naturally occurring RNA through the addition, deletion, substitution, and/or modification of one or more nucleotides. The nucleotides in the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, as well as inert components. The use of such pharmaceutically acceptable carriers or excipients for the active pharmaceutical ingredient is well known in the art. Unless any conventionally pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is covered. Additional active pharmaceutical ingredients, such as those of other pharmaceuticals, may also be incorporated into the described compositions and methods.

The terms “about” and “approximately” mean a range of values that are statistically significant. This range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The permissible differences covered by the terms “about” or “approximately” depend on the specific system under study and are readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not precise and do not need to be precise, but may be approximate and/or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. Generally, whether so explicitly stated or not, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are “about” or “approximately”. It should be noted that embodiments with very different sizes, shapes, and dimensions may employ the described arrangement.

When used in the appended claims in both original and modified forms, the transitional terms “comprising,” “consisting essentially of,” and “consisting of” define the scope of the invention with respect to which additional, unrecited elements or steps (if present) are excluded from the scope of the claims. The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case excludes impurities generally associated with the specified materials. The term “consisting essentially of” limits the scope of the invention to the specified elements, steps, or materials and those elements, steps, or materials that do not substantially affect the basis and novel features of the claimed invention. In alternative embodiments, all compositions, methods, and kits for practicing the invention described herein may be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

The term “antibody” and its plural form “antibodies” refer to a complete immunoglobulin and any antigen-binding fragment (“antigen-binding moiety”) or its single chain. “Antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or its antigen-binding moiety. Each heavy chain includes a heavy chain variable region (abbreviated as _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated as _VL ) and a light chain constant region. The light chain constant region comprises one domain, _CL . The _VH and _VL regions of an antibody can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), and these may be interspersed with more conserved regions called frame regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more epitopes. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen" refers to a substance that induces an immune response. In some embodiments, if presented via a major histocompatibility complex (MHC) molecule, the antigen is a molecule capable of being bound by an antibody or TCR. As used herein, the term "antigen" also encompasses T-cell epitopes. Antigens are additionally capable of being recognized by the immune system. In some embodiments, the antigen is capable of inducing a humoral or cellular immune response that results in the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require the antigen to contain or be linked to a Th-cell epitope. The antigen may also have one or more epitopes (e.g., B epitopes and T epitopes). In some embodiments, the antigen is preferably to react generally with its corresponding antibody or TCR in a highly specific and selective manner, and not with a variety of other antibodies or TCRs that can be induced by other antigens.

The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to formulations of antibody molecules that are single molecular compositions. Monoclonal antibody compositions exhibit single binding specificity and affinity against a specific epitope. Monoclonal antibodies specific to certain receptors can be prepared using knowledge and techniques in the following methods: injecting a suitable antigen into a test subject and then isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. DNA encoding monoclonal antibodies can be isolated and sequenced using routine procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not normally produce immunoglobulins (e.g., *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to obtain monoclonal antibody synthesis in recombinant host cells. The recombinant production of antibodies will be described in more detail below.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Antigen-binding function of antibodies has been demonstrated to be performed by fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of _VL , _VH , _CL , and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; (iii) Fd fragments consisting of _VH and CH1 domains; (iv) Fv fragments consisting of _VL and _VH domains of a single arm of an antibody; (v) domain antibody (dAb) fragments (Ward et al., Nature, 1989, 341, 544-546), which may consist of a _VH or a _VL domain; and (vi) segregated complementarity-determining regions (CDRs). Furthermore, although the two domains ( _VL and _VH ) of the Fv fragment are encoded by independent genes, they can be joined using recombination methods via a synthetic adapter that enables the creation of a single protein chain (referred to as a single-chain Fv (scFv)) in which the _VL and _VH regions pair to form a monovalent molecule; see, for example, Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are targeted at the utility screening fragment in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises both the _VH and _VL portions. A scFv molecule is represented as _VL - _LVH when the _VL domain is the N-terminal portion of the scFv molecule, or as _VH - _LVL when the _VH domain is the N-terminal portion of the scFv molecule. Methods for preparing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB, Vol. 9: 73-80 (1995); and REBird and BWWalker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9: 132-137 (1991), the disclosures of which are incorporated herein by reference.

As used herein, the term "human antibody" is intended to include antibodies that have variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, said constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human framework sequence.

The term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity with a variable region, wherein both the framework and CDR regions of the variable region are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is generated from a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with immortalized cells, having a genome containing both human heavy-chain and light-chain transgenes.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas prepared therefrom (described further below) that are transgenic for the human immunoglobulin gene; (b) antibodies isolated from host cells transformed to express human antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may undergo in vitro mutation induction (or in vivo somatic mutation induction when using transgenic animals with human Ig sequences), and therefore the amino acid sequences of the _VH and _VL regions of the recombinant antibody, although derived from and associated with human germline _VH and _VL sequences, may not be naturally present in the human antibody germline in vivo.

As used in this article, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

The term "human antibody derivative" refers to any modified form of a human antibody, including conjugates of antibodies with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which may be conjugated to the antibody described herein using methods available in the art.

The terms "humanized antibody" and "humanized" refer to antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence. Further frame region modifications can be made to the human frame sequence. Humanized forms of non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. To a large extent, humanized antibodies are human immunoglobulins (receptor antibodies) where residues from the hypervariable region of the receptor are replaced by residues from 15 hypervariable regions of a non-human species (donor antibody) possessing the desired specificity, affinity, and ability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in receptor or donor antibodies. These modifications are performed to further optimize antibody performance. Generally, humanized antibodies will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially all FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein may also be modified to use any Fc variant known to provide effector function and/or FcR binding improvements (e.g., reductions). Fc variants may include any of the amino acid substitutions disclosed below: International Patent Application Publications No. WO 1988/07089A1, WO 1996/14339A1, WO 1998/05787A1, WO 1998/23289A1, WO 1999/51642A1, WO 99/58572A1, WO 2000/09560A2, WO 2000/32767A1, WO 20 No. 00/42072A2, No. WO 2002/44215A2, No. WO 2002/060919A2, No. WO 2003/074569 A2, No. WO 2004/016750 A2, No. WO 2004/029207 A2, No. WO 2004/035752 A2, No. WO 2004/063351A2, No. WO 2004/074455 A2, No. WO 2004/09924 9 A2, WO 2005/040217 A2, WO 2005/070963 A1, WO 2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 A1, WO 2006/047350 A2 and WO 2006/085967 A2; and U.S. Patent No. 5,648,260; Nos. 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the contents of which are incorporated herein by reference.

The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

A “bifunctional antibody” is a small antibody fragment having two antigen-binding sites. The fragment contains a heavy chain variable domain (V_{_H} ), which is linked to a light chain variable domain (V_L ) in the same polypeptide chain (V_{_H}-V_{_L} or V_{_L}-V_{_H}). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, thus creating two antigen-binding sites. Bifunctional antibodies are described in more detail, for example, in European Patent No. 404,097, International Patent Publication No. WO 93/11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

The term "glycosylation" refers to a modified derivative of an antibody. Deglycosylated antibodies are not glycosylated. Glycosylation can be altered to, for example, increase the antibody's affinity for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at said sites. Deglycosylation can increase the antibody's affinity for an antigen, as described in U.S. Patents 5,714,350 and 6,350,861. Alternatively or additionally, antibodies with altered glycosylation types can be produced, such as low-fucosylated antibodies with reduced fucosylation residues or antibodies with increased fractional GlcNac structure. Such altered glycosylation patterns have been shown to enhance antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention to produce glycosylated-modified antibodies. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)fucosyltransferase), so that antibodies expressed in Ms704, Ms705, and Ms709 cell lines do not have fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/- cell lines were formed by breaking the FUT8 gene in targeted CHO/DG44 cells using two alternative vectors (see, for example, U.S. Patent Publication No. 2004/0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation by reducing or eliminating α1,6 bond-related enzymes. The patent also describes cell lines with low or no enzymatic activity for adding fucose to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). International Patent Publication WO 03/035835 describes a variant CHO cell line, namely Lec 13 cells, which has a reduced ability to link fucose to Asn(297)-linked carbohydrates and also induces hypofucosylation of antibodies expressed in said host cells (see also Shields et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication WO 99/54342 describes cells engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine). Cell lines containing GnTIII transferase III (GnTIII) can be used to enhance the antibody's ADCC activity by causing antibodies expressed in engineered cell lines to exhibit an increased bipartite GlcNac structure (see also Umana et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidases can be used to cleave the fucosylate residues of the antibody. For example, α-L-fucosidase removes fucosylate residues from the antibody, as described in Tarentino et al., Biochem. 1975, 14, 5516-5523.

“Polyglycolization” refers to the modification of an antibody or fragment thereof, typically with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that link one or more PEG groups to the antibody or antibody fragment. For example, PEGylation can increase the biological (e.g., serum) half-life of an antibody. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to cover any form of PEG that has been used to derive other proteins, such as mono( _C1 - _C10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-cis-butenediamide. The antibody to be PEGylated is a deglycosylated antibody. Polyethylene glycolation methods are known in the art and applicable to antibodies used in this invention, such as those described, for example, in European Patents EP0154316 and EP0401384 and U.S. Patent No. 5,824,778, the disclosures of which are each incorporated herein by reference.

The term "biosimilar" refers to a biological product (including monoclonal antibodies or proteins) that meets the following criteria: it is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, and there are no clinically significant differences between the biological product and the reference product in terms of safety, purity, and efficacy. Furthermore, a biosimilar or "biosimilar" drug is a biological drug similar to another biological drug authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. A biological product or biological drug is a drug derived from or produced from a biological source (e.g., bacteria or yeast). It may consist of relatively small molecules (e.g., human insulin or erythropoietin) or complex molecules (e.g., monoclonal antibodies). For example, if the reference IL-2 protein is proleukin, then the protein of the reference proleukin approved by a drug regulatory agency is "biosimilar to proleukin" or "a biosimilar of proleukin." In Europe, a biosimilar or “biosimilar” medicine is a biological medicine similar to another biological medicine that has been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for the use of biosimilars in Europe is Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended, and therefore in Europe, biosimilars may be authorized, approved for authorization, or submitted as the subject of authorization pursuant to Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC. The original biological medicine product authorized in Europe may be referred to as a “reference medicine.” The CHMP guidance on biosimilar products outlines some of the requirements for a product to be considered a biosimilar. In addition, product-specific guidance, including guidance relating to monoclonal antibody biosimilars, is provided by the EMA on a product-by-product basis and published on its website. Biosimilars as described herein may be similar to a reference medicine in terms of quality characteristics, biological activity, mechanism of action, safety profile, and/or efficacy. Furthermore, biosimilars may be used or intended for the treatment of the same disease as the reference medicine. Therefore, biosimilars as described herein can be considered to have similar or very similar quality characteristics to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar biological activity to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar safety profile to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar efficacy to the reference drug. As described herein, in Europe, biosimilars have been compared with reference drugs authorized by the EMA. However, in some cases, in certain studies, biosimilars may be compared with biological products authorized outside the European Economic Area (non-EEA authorized "comparatives"). Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to biological products that have been or may be compared with non-EEA authorized comparatives. Some biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding moieties), and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications (including, for example, amino acid deletions, additions, and/or substitutions) in their amino acid structure that do not significantly affect peptide function. Biosimilars may contain an amino acid sequence having 97% or higher, such as 97%, 98%, 99%, or 100%, sequence identity with the amino acid sequence of a reference drug. Biosimilars may contain one or more post-translational modifications that differ from those of the reference drug, such as (but not limited to) glycosylation, oxidation, deamidation, and/or truncation, provided that such differences do not alter the safety and/or efficacy of the drug. Biosimilars may have the same or different glycosylation patterns as the reference drug. In particular, but not exclusively, biosimilars may have different glycosylation patterns if such differences resolve or are intended to resolve safety concerns associated with the reference drug. Additionally, biosimilars may differ from the reference drug in, for example, their potency, pharmaceutical form, formulation, excipients, and/or delivery method, provided that this does not impair the safety and efficacy of the drug. Compared to a reference product, biosimilars may contain differences in, for example, pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles, but are still considered sufficiently similar to the reference product and thus eligible for or suitable for authorization. In some cases, biosimilars exhibit different binding characteristics compared to the reference product, where the regulatory agency (e.g., the EMA) does not consider these different binding characteristics as an obstacle to authorization of the biosimilar product. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions.

II. Gene Editing Process

A. Overview: TIL amplification + gene editing

Embodiments of the present invention relate to methods for expanding a TIL population, said methods comprising one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, “gene-editing” and “genome editing” refer to a gene modification in which DNA is permanently modified in the genome of a cell, such as by inserting, deleting, modifying, or replacing DNA within the cell's genome. In some embodiments, gene editing causes silencing (sometimes referred to as gene knockout) or suppression/reduction (sometimes referred to as gene knockdown) of DNA sequence expression. According to embodiments of the present invention, gene editing technology is used to enhance the effectiveness of a therapeutic TIL population.

Methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the methods further include gene editing of at least a portion of the TILs. According to other embodiments, methods for expanding TILs into a therapeutic TIL population can be carried out according to any embodiment of the methods described in WO 2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, which are incorporated herein by reference in their entirety, wherein the methods further include gene editing of at least a portion of the TILs. Therefore, one embodiment of the invention provides a therapeutic TIL population expanded according to any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, for example, at least a portion of the therapeutic TIL population transferred to an infusion bag has been permanently gene-edited.

Gene editing during B.TIL amplification

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the method includes the step of culturing or initially expanding a first TIL population, comprising culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some embodiments, the method includes the steps of culturing or rapidly expanding the third TIL population by culturing the third TIL population in a second cell culture medium for a first period of about 1-7 days, splitting the culture into multiple subcultures at the end of the first period, culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 3-7 days, and merging the multiple subcultures at the end of the second period to provide an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the activation of the second TIL population takes approximately 7 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during, or before, any of steps (a)-(e), (a)-(f), or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e), (a)-(f), or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A third population of TILs was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some embodiments, the step of culturing the third TIL population takes approximately 7 days. In some embodiments, the step of culturing the third TIL population takes approximately 8 days. In some embodiments, the step of culturing the third TIL population takes approximately 9 days. In some embodiments, the step of culturing the third TIL population takes approximately 10 days. In some embodiments, the step of culturing the third TIL population takes approximately 11 days. In some embodiments, the step of culturing the third TIL population takes approximately 12 days. In some embodiments, the step of culturing the third TIL population takes approximately 13 days. In some embodiments, the step of culturing the third TIL population takes approximately 14 days. In some embodiments, the step of culturing the third TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(d) or (a)-(e) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(d) or (a)-(e), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some implementation schemes, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementation schemes, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are then placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 1-7 days. In some implementations, the step of culturing the third TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the third TIL population takes approximately 1 day. In some implementations, the step of culturing the third TIL population takes approximately 2 days. In some implementations, the step of culturing the third TIL population takes approximately 3 days. In some implementations, the step of culturing the third TIL population takes approximately 4 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some implementations, the process of culturing a third TIL population takes about 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed over a period of approximately 22 days. In some embodiments, the steps of the method are completed over a period of approximately 23 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some implementations, the steps of the method are completed over a period of approximately 22 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1 day. In some implementations, the step of culturing the fourth TIL population takes approximately 2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 3 days. In some implementations, the step of culturing the fourth TIL population takes approximately 4 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, initial amplification takes approximately 3-9 days. In other implementations, initial amplification takes approximately 1-9 days, 2-9 days, 3-9 days, approximately 4-9 days, approximately 5-9 days, approximately 6-9 days, approximately 7-9 days, approximately 8-9 days, approximately 1-8 days, approximately 2-8 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, initial amplification takes approximately 1 day. In some embodiments, initial amplification takes approximately 2 days. In some embodiments, initial amplification takes approximately 3 days. In some embodiments, initial amplification takes approximately 4 days. In some embodiments, initial amplification takes approximately 5 days. In some embodiments, initial amplification takes approximately 6 days. In some embodiments, initial amplification takes approximately 7 days. In some embodiments, initial amplification takes approximately 8 days. In some embodiments, initial amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the rapid second amplification takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error or incomplete sentence.) The durations of rapid secondary amplification are approximately 11-13 days, 12-13 days, 5-12 days, 6-12 days, 7-12 days, 8-12 days, 9-12 days, 10-12 days, 11-12 days, 5-11 days, 6-11 days, 7-11 days, 8-11 days, 9-11 days, 10-11 days, 5-10 days, 6-10 days, 7-10 days, 8-10 days, 9-10 days, 5-9 days, 6-9 days, 7-9 days, 8-9 days, 5-8 days, 6-8 days, 7-8 days, 5-7 days, 6-7 days, and 5-6 days. In some implementations, rapid secondary amplification takes approximately 5 days. In some implementations, rapid secondary amplification takes approximately 6 days. In some implementations, rapid secondary amplification takes approximately 7 days. In some embodiments, rapid second amplification takes approximately 8 days. In some embodiments, rapid second amplification takes approximately 9 days. In some embodiments, rapid second amplification takes approximately 10 days. In some embodiments, rapid second amplification takes approximately 11 days. In some embodiments, rapid second amplification takes approximately 12 days. In some embodiments, rapid second amplification takes approximately 13 days. In some embodiments, rapid second amplification takes approximately 14 days. In some embodiments, rapid second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digest the tumor tissue sample or tumor fragment in an enzyme medium to produce tumor digest;

(c) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some implementations, the first amplification takes approximately 3-9 days. In some implementations, the first amplification takes approximately 3-9 days, approximately 3-8 days, approximately 3-7 days, approximately 3-6 days, approximately 3-5 days, approximately 3-4 days, approximately 4-9 days, approximately 4-8 days, approximately 5-9 days, approximately 5-8 days, approximately 6-9 days, approximately 6-8 days, approximately 7-9 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the first amplification takes approximately 3 days. In some implementations, the first amplification takes approximately 4 days. In some implementations, the first amplification takes approximately 5 days. In some implementations, the first amplification takes approximately 6 days. In some implementations, the first amplification takes approximately 7 days. In some implementations, the first amplification takes approximately 8 days. In some implementations, the first amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes approximately 5-15 days. In some implementations, the second amplification takes approximately 5-15 days, approximately 6-15 days, approximately 7-15 days, approximately 8-15 days, approximately 9-15 days, approximately 10-15 days, approximately 11-15 days, approximately 12-15 days, approximately 13-15 days, approximately 14-15 days, approximately 5-14 days, approximately 6-14 days, approximately 7-14 days, approximately 8-14 days, approximately 9-14 days, approximately 10-14 days, approximately 11-14 days, approximately 12-14 days, approximately 13-14 days, approximately 5-13 days, approximately 6-13 days, approximately 7-13 days, approximately 8-13 days, approximately 9-13 days, approximately 10-13 days. The durations of amplification are as follows: approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the second amplification takes approximately 5 days. In some implementations, the second amplification takes approximately 6 days. In some implementations, the second amplification takes approximately 7 days. In some implementations, the second amplification takes approximately 8 days. In some embodiments, the second amplification takes approximately 9 days. In some embodiments, the second amplification takes approximately 10 days. In some embodiments, the second amplification takes approximately 11 days. In some embodiments, the second amplification takes approximately 12 days. In some embodiments, the second amplification takes approximately 13 days. In some embodiments, the second amplification takes approximately 14 days. In some embodiments, the second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days. In some embodiments, the steps of the method are completed within a period of approximately 32 days.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

In some implementations, gene editing is performed while the TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementations, TILs collected from the culture medium are gene-edited, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, the step of gene editing at least a portion of the second or third TIL population includes a sterile electroporation step on the second or third TIL population.

In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some embodiments, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step further includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 22 hours. According to some implementations, the quiescent step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the antigen-presenting cells (APCs) are PBMCs. According to some embodiments, the PBMCs are irradiated. According to some embodiments, the PBMCs are allogeneic. According to some embodiments, the PBMCs are both irradiated and allogeneic. According to some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some implementations, the tumor tissue is derived from an autopsied tumor.

In some implementations, the dissected tumor is less than 8 hours old.

In some embodiments, the tumor tissue is selected from the group consisting of: melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple-negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and HPV-positive tumor tissue.

In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 2 mm.

In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 1.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5.5 mm and a longest side length of about 6 mm.

In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm or about 6 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 6 mm.

In some embodiments, the present invention provides a therapeutic population of tumor-infiltrating lymphocyte (TIL) products generated by methods as described herein.

In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising administering to the patient an effective amount of a therapeutic TIL population generated by the method described herein. In some embodiments, the cancer is selected from the group consisting of: glioblastoma (GBM), gastrointestinal cancer, melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, and mantle cell lymphoma. In some implementations, the cancer is selected from the group consisting of: cutaneous melanoma, ocular melanoma, uveal melanoma, conjunctival malignant melanoma, metastatic melanoma, pleomorphic xanthoastrocytoma, dysembryonic neuroepithelial tumor, ganglioglioma and pilocytic astrocytoma, endometrioid adenocarcinoma with significant myxoid differentiation (ECMD), papillary thyroid carcinoma, serous low-grade or borderline ovarian cancer, hairy cell leukemia and Langerhans cell histiocytosis.

In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 2000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 2000 IU/mL.

In some embodiments, in the second amplification step, the IL-2 is present at an initial concentration between 1000 IU/mL and 6000 IU/mL, and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some embodiments, the first amplification is performed using a ventilated container. In some embodiments, the second amplification is performed using a ventilated container.

In some embodiments, the first cell culture medium further comprises cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and/or the third culture medium further comprise cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the method further includes the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated the day after administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated on the same day as administration of the TIL or PBL product. In some embodiments, the IL-2 regimen comprises adelleukin, nevoleukin, or a bioanalyte or variant thereof.

In some embodiments, the therapeutically effective amount of TIL product comprises about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs.

In some implementations, the number of the second TIL population is at least 50 times that of the first TIL population.

C.PD-1

One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 superfamily of T cell regulators. Its ligands, PD-L1 and PD-L2, are expressed on various tumor cells, including melanoma. The interaction between PD-1 and PD-L1 can suppress T cell effector function, cause T cell exhaustion under chronic stimulation, and induce T cell apoptosis in the tumor microenvironment. PD-1 may also play a role in tumor-specific evasion of immune surveillance.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of PD-1 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the method includes gene editing of at least a portion of the TILs by silencing or inhibiting the expression of PD-1. As described in more detail below, the gene editing process may involve the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., PD-1). For example, the TALEN method can be used to silence or reduce the expression of PD-1 in TILs.

D.CTLA-4

CTLA-4 expression is induced upon T cell activation on activated T cells and competitively binds to antigen-presenting cell activation antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 can induce T cell suppression and is used to maintain the homeostasis of the immune response. However, inhibiting the interaction of CTLA-4 with CD80 or CD86 prolongs T cell activation and thus increases the level of the immune response against cancer antigens.

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of CTLA-4 in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and CTLA-4 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CTLA-4. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CTLA-4). For example, the CRISPR method, the TALE method, or the zinc finger method can be used to silence or inhibit the expression of CTLA-4 in TILs. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and CTLA-4 in TILs.

E.LAG-3

Following conjugation at the major histocompatibility complex (MHC) class II, lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells. Although the mechanism is not fully understood, regulation of this gene can induce negative regulation of T cell function, preventing tissue damage and autoimmunity. Therefore, LAG-3 blockade may improve antitumor responses. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of LAG-3 in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and LAG-3 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of LAG-3. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., LAG-3). According to specific embodiments, the expression of LAG-3 in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and LAG-3 in TILs can be silenced or reduced using the TALEN method.

F.Cish

Cish, a member of the cytokine signaling inhibitory factor (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits its functional affinity for tumors. Deletion of the Cish gene in CD8+ T cells enhances its amplification, functional affinity, and cytokine pluripotency, leading to significant and durable regression of existing tumors. See, for example, Palmer et al., Journal of Experimental Medicine, 212(12):2095 (2015).

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of Cish in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and Cish in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of Cish. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., Cish). For example, the expression of Cish in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and Cish in TILs can be silenced or reduced using the TALEN method.

G.CBL-B

CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and a negative regulator of T cell activation. (Bachmaier et al., Nature, 2000, 403, 211-216; Wallner et al., Clin. Dev. Immunol. 2012, 692-639.)

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of CBL-B in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and CBL-B in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., process 2A or the method shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CBL-B. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CBL-B). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, CBL-B is silenced using TALEN gene knockout. In some embodiments, CBL-B is silenced using a TALE-KRAB transcription inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and CBL-B in TIL.

H.TIGIT

TIGIT is a cell surface protein expressed on regulatory, memory, and activated T cells. TIGIT belongs to the poliovirus receptor (PVR) family of immunoglobulins and inhibits T cell activation. (Yu et al., Nat Immunol., 2009, 10(1):48-57).

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of TIGIT in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and TIGIT in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of TIGIT. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., TIGIT). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, TIGIT can be silenced using TALEN gene knockout. In some embodiments, TIGIT can be silenced using a TALE-KRAB transcriptional inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and TIGIT in TIL.

III. Gene Editing Methods

As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) genetically modified via gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing performed via nucleotide insertion (RNA or DNA) into a TIL population to suppress the expression of one or more proteins. Embodiments of the present invention also provide a method for expanding TILs into a therapeutic population, wherein the method includes gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, and these gene editing techniques are suitable for use according to the present invention.

In some embodiments, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes a retroviral transduction step. In one embodiment, the method of genetically modifying a TIL population includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, a method for genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which a transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long-term expression of the transposase does not occur in transgenic cells, for example, a transposase provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems include salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In one embodiment, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing or repressing (e.g., silencing) one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In another embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating TILs with a pulsed electric field to induce pore formation in the TILs, including the step of applying a series of at least three DC electrical pulses to the TILs, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time and such that the survival rate of the TILs is maintained. In one embodiment, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes the step of transfection using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

According to one embodiment, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems, such as Cas9, target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA bases and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, a method for amplifying TILs into a therapeutic population may be performed according to any embodiment of the method described herein (e.g., procedure 2A) or as described in WO2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, wherein said method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by comparing gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

1. TALE method

A method for amplifying TILs into a therapeutic population can be made according to any embodiment of the method described herein or as described in WO 2018/081473 A1, WO 2018/129332A1, or WO 2018/182817 A1, wherein the method further includes gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the use of the TALE method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the TALE method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for “transcription activator-like effector” protein, which includes TALEN (“transcription activator-like effector nuclease”). The method of gene editing using the TALE system is also referred to herein as the TALE method. TALE is a naturally occurring protein from the plant pathogenic bacterium *Xanthomonas* and contains a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize continuous DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by 14–20 base-pair spacers, bring FokI monomers closer together for dimerization and to generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and T NFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA , IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

Non-limiting examples of TALE-nucleases targeting the PD-1 gene are provided in the table below. In these examples, the targeted genomic sequence contains two 17-base-pair (bp) long sequences (referred to as half-targets, shown in uppercase letters) separated by a 15-bp spacer. Each half-target is recognized by a repetitive sequence of the half-TALE-nuclease listed in the table. Thus, according to a particular embodiment, the TALE-nuclease according to the invention recognizes and cleaves target sequences selected from the group consisting of: SEQ ID NO:127 and SEQ ID NO:128. TALEN sequences and gene editing methods are also described in Gautron et al., Molecular Therapy: Nucleic Acids, December 2017, Vol. 9: 312-321, which is incorporated herein by reference.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

2. Cas-CLOVER method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs using a Cas-CLOVER method. According to a particular embodiment, the use of the Cas-CLOVER method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the Cas-CLOVER method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Cas-CLOVER is a dimerizing, high-fidelity site-specific nuclease (SSN) composed of a fusion of catalytically dead SpCas9 (dCas9) and a nuclease domain from a Clostridium Clo051 type IIs restriction endonuclease (Madison et al., “Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched alloeneic CAR-T cells”, Molecular Therapy-Nucleic Acids, 2022). This produces a nuclease whose activity is determined by the dimerization of the Clo051 nuclease domain, which recognizes two adjacent 20-nt target sequences via RNA guidance. Unlike paired nicking enzyme approaches, for example, when using the Cas9-D10A mutant, monomeric Cas-CLOVER does not introduce nicks or DSBs. Cas-CLOVER has been shown to have low off-target nuclease activity.

Exemplary Cas-CLOVER systems include those described in WO2019/126578, the contents of which are incorporated herein by reference in their entirety. In embodiments, the Cas-CLOVER system comprises a fusion protein containing a DNA-localizing component and an effector molecule, substantially consisting of or consisting of therein.

DNA positioning components

In this embodiment, the DNA targeting component is capable of binding to a specific DNA sequence. In this embodiment, the DNA targeting component is selected from, for example, DNA-binding oligonucleotides, DNA-binding proteins, DNA-binding protein complexes, and combinations thereof. Other suitable DNA-binding components will be recognized by those skilled in the art.

In one embodiment, the DNA targeting component comprises oligonucleotides targeting one or more specific loci in the genome. The oligonucleotides may be selected from DNA, RNA, DNA/RNA hybrids, and combinations thereof.

In some embodiments, the DNA positioning component comprises a nucleotide-binding protein or protein complex that binds an oligonucleotide upon binding to the target DNA. The protein or protein complex may be able to recognize features selected from RNA-DNA heteroduplexes, R-loops, or combinations thereof. In some embodiments, the DNA positioning component comprises a protein or protein complex capable of recognizing an R-loop selected from Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, or combinations thereof. In some embodiments, the DNA positioning component comprises an engineered protein capable of binding to the target DNA. In some embodiments, the DNA positioning component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, transcription activator-like (TAL) arrays, and combinations thereof. In some embodiments, the DNA positioning component comprises a protein containing a naturally occurring DNA-binding domain. In some embodiments, the DNA positioning component comprises a bZIP domain, a helix-loop-helix, a helix-turn-helix, an HMG-box, a leucine zipper, a zinc finger, or combinations thereof. In some embodiments, the DNA positioning component comprises an oligonucleotide targeting a specific locus in the genome. Exemplary oligonucleotides include, but are not limited to, DNA, RNA, DNA/RNA hybrids, and any combination thereof. In some embodiments, the DNA targeting component comprises a protein or protein complex capable of recognizing features selected from RNA-DNA heteroduplexes, R-loops, and any combinations thereof. Exemplary proteins or protein complexes capable of recognizing R-loops include, but are not limited to, Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, and any combinations thereof. In some embodiments, the protein or protein complex capable of recognizing R-loops comprises Cas9. In some embodiments, the DNA targeting component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, TAL arrays, and any combinations thereof. In some embodiments, the DNA targeting component comprises an oligonucleotide targeting a specific location in the genome and a protein capable of binding to a target DNA sequence.

In one embodiment, the DNA positioning component comprises at least one guide RNA (gRNA), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises two gRNAs, substantially consisting of or consisting of them, wherein the first gRNA specifically binds to the first strand of the double-stranded DNA target sequence, and the second gRNA specifically binds to the second strand of the double-stranded DNA target sequence. Alternatively, in one embodiment, the DNA positioning component comprises a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Ralstonia*, substantially consisting of or consisting of it.

Effect molecules

In the implementation scheme, the effector molecule is capable of producing a predetermined effect at a specific locus in the genome. Exemplary effector molecules include, but are not limited to, transcription factors (activators or repressors), chromatin remodeling factors, nucleases, exonucleases, endonucleases, transposases, methyltransferases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, helicases, fluorophores, or any combination thereof.

In some embodiments, the effector molecule comprises a transposase. In some embodiments, the effector molecule comprises PB transposase (PBase). In some embodiments, the effector molecule comprises a nuclease. Non-limiting examples of nucleases include restriction endonucleases, homing endonucleases, S1 nucleases, mung bean nucleases, pancreatic DNase I, micrococcal nucleases, yeast HO endonucleases, or any combination thereof. In some embodiments, the effector molecule comprises a restriction endonuclease. In some embodiments, the effector molecule comprises a type IIS restriction endonuclease. In some embodiments, the effector molecule comprises a nuclease. Non-restrictive examples of endonucleases include AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, and Clo051. In the implementation scheme, the effector molecule includes BmrI, BfiI, or Clo051.

In embodiments, the effector molecule comprises a homodimer or heterodimer, substantially composed of, or composed of. In embodiments, the effector molecule comprises a nuclease, optionally an endonuclease, substantially composed of, or composed of. In embodiments, the effector molecule, including those comprising a homodimer or heterodimer, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially composed of, or composed of. In embodiments, Cas9 is catalytically inactive or “inactivated” Cas9 (dCas9 (SEQ ID NO: 302 and 303 of WO2019/126578)). In embodiments, Cas9 is the catalytically inactive or “inactivated” nuclease domain of Cas9. In embodiments, dCas9 is encoded by a shorter sequence derived from full-length, catalytically inactivated Cas9, referred herein as “small” dCas9 or dSaCas9 (SEQ ID NO: 23 of WO2019/126578).

In embodiments of the fusion protein, the effector molecule comprises, is substantially composed of, or is composed of, one or more type II nucleases, homodimers or heterodimers of type II nucleases. In the implementation scheme, the type II nuclease includes one or more of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof that forms a heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises a catalytically inactive form of Cas9 (e.g., dCas9 or dSaCas9) or a fragment thereof that forms a heterodimer with Clo051, and is substantially composed of or constitutes thereof. An exemplary Clo051 nuclease domain may comprise the following amino acid sequence, and is substantially composed of or constitutes thereof:

EGIKSNISLLKDELRGQISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE

KIRSGEMTIEELERAMFNNSEFILKY(SEQ ID NO:238).

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces*, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, and is substantially composed of or constitutes thereof.

connect

In some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule, substantially consisting of or consisting of the latter. In some embodiments, nucleic acid sequences encoding one or more components of the fusion protein can be operatively linked, for example, to an expression vector. In some embodiments, the fusion protein is a chimeric protein. In some embodiments, the fusion protein is encoded by one or more recombinant nucleic acid sequences. In some embodiments, the fusion protein further includes a linker region to operatively link the two components of the fusion protein. For example, in some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule operatively linked by a linker region, substantially consisting of or consisting of the latter. In some embodiments, the DNA positioning component, linker region, and effector molecule can be encoded by one or more nucleic acid sequences inserted into an expression cassette and/or expression vector, such that translation of the nucleic acid sequences produces the fusion protein. In some embodiments, the fusion protein can include a non-covalent link between the DNA positioning component and the effector molecule. The non-covalent link can comprise an antibody, antibody fragment, antibody mimic, or scaffold protein.

Fusion protein

In one embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated Cas9 (dCas9) or an inactivated nuclease domain, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated small Cas9 (dSaCas9), substantially consisting of or consisting of it. In yet another embodiment, the effector molecule comprises Cas9, dCas9, dSaCas9, or a nuclease domain thereof and a second endonuclease, substantially consisting of or consisting of it. The second endonuclease may include, or be substantially composed of, type IIS endonucleases, including but not limited to, AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, and BmgBI. One or more of BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI, or Clo051.

In embodiments of the fusion protein, the DNA localization component comprises, is substantially composed of, or is composed of, a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), and the effector molecule comprises, substantially composed of, or is composed of, a nuclease. In embodiments of the fusion protein of this disclosure, the DNA localization component comprises, substantially composed of, or is composed of, a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rhodotorula*, and the effector molecule comprises, substantially composed of, or is composed of, an IIS-type nuclease, including but not limited to AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My One or more of I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In some embodiments, the exemplary dCas9-Clo051 fusion protein may comprise, substantially consist of, or consist of the amino acid sequence of SEQ ID NO:305 or 307 of WO2019/126578 or the nucleic acid sequence of SEQ ID NO:306 or 308 of WO2019/126578.

Construct

In the embodiments, the nuclease domain comprises dCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises dSaCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Xanthomonas spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Rocheborax spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the fusion protein comprises dCas9-Clo051, dSaCas9-Clo051, Xanthomonas spp.-TALE-Clo051, or Rocheborax spp.-TALE-Clo051. In the embodiments, the vector encoding the fusion protein comprises Csy4-T2A-Clo051-G4S linker-dCas9 (Streptococcus pyogenes) or pRT1-Clo051-dCas9 dual NLS.

According to some implementations, the Cas-CLOVER system comprises a fusion protein containing a DNA localization component and an effector molecule, wherein the DNA localization component hybridizes to a target sequence of a DNA molecule in a TIL, wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule, and the effector molecule cleaves the DNA molecule, thereby altering the expression of at least one immune checkpoint molecule.

According to a specific implementation, the Cas-CLOVER method involves silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas-CLOVER system (e.g., dCas9-Clo051, dSaCas9-Clo051, Xanthomonas-TALE-Clo051, or Rochetomyces-TALE-Clo051 fusion protein) that is specific to the target DNA sequence of immune checkpoint genes. The fusion protein can be delivered as DNA, mRNA, or protein. After the genome comes into contact with the Cas-CLOVER system, one or more strands of the target double-stranded DNA can be cleaved. If cleavage occurs in the presence of one or more DNA repair pathways or components thereof, the Cas-CLOVER method interrupts gene expression or modifies the genomic sequence by inserting, deleting, or substituting one or more base pairs. DSBs in cells can be repaired via non-homologous end joining (NHEJ, a mechanism that typically causes insertions or deletions (insertion/deletion) in DNA). Insertions/deletions typically induce frameshifts, resulting in loss-of-function alleles; for example, by inducing premature stop codons within the open reading frame (ORF) of a target gene. According to some implementations, the result is a loss-of-function mutation within a target immune checkpoint gene.

Alternatively, instead of NHEJ, DSB induced by the Cas-CLOVER system can be repaired via homology-directed repair (HDR). Although NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to produce specific nucleotide changes ranging from a single nucleotide change to a large insertion range. According to some implementations, HDR is used to gene edit immune checkpoint genes by delivering a DNA repair template containing the desired sequence to a TIL equipped with a Cas-CLOVER system. The repair template preferably contains the desired edit immediately upstream and downstream of the target gene, as well as other homologous sequences (often referred to as left and right homologous arms).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the Cas-CLOVER method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF1. 0A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, I L6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via the Cas-CLOVER method and which can be used according to embodiments of the invention are described in WO2019126578, US2017/0107541, US2017/0114149, US2018/0187185, and U.S. Patent No. 10,415,024, the contents of which are incorporated herein by reference in their entirety. Resources for performing the Cas-CLOVER method, such as CLOVER mRNA and Cas-CLOVER mRNA constructs, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system regulating the expression of at least one checkpoint protein in multiple cells of a second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culture for about 1 to 3 days to obtain the second TIL population, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system inhibiting the expression of at least one checkpoint protein in multiple cells of the second TIL population.

IV. Gene Expression Methods

In some embodiments, the method of genetically modifying TIL populations to regulate protein expression may optionally include a step of stabilizing and incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying TIL populations includes a viral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a gamma-retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adenovirus transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adeno-associated virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a herpes simplex virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a poxvirus transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step, including lentiviral transduction using human immunodeficiency virus (HIV) (including HIV-1). Lentiviral transduction systems and other suitable viral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat’l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71, and U.S. Patents 5,350,674, 5,585,362, and 6,627,442, the disclosures of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16; Hawley et al., Gene Ther. 1994, 1, 136-38; the contents of which are incorporated herein by reference.

1. piggyBac method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs by a piggyBac method (e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases). According to a particular embodiment, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population. Alternatively, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population and optionally induce the enhancement of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. In some embodiments, at least one immunomodulatory composition comprises an immunomodulator fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and CD40 agonists (e.g., CD40L or agonistic CD40 binding domains). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and CD40 agonists. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and CD40 agonists.

The piggyBac transposon is a mobile genetic element that efficiently translocates between the donor vector and the host chromosome. This system has virtually no cargo limitations and is completely reversible, leaving no footprint in the genome after excision. The piggyBac transposon/transposase system consists of transposases that recognize piggyBac-specific inverted terminal repeats (ITRs) located flanking the transposon cassette. The transposases excise the transposable element to integrate it into a TT/AA chromosomal locus that preferentially resides in euchromatin regions of the mammalian genome (Ding et al., 2005; and Bradley 2007; Wilson et al., 2007; Wang et al., 2008; Li et al., 2011).

Exemplary piggyBac systems include those described in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac system comprises a transposon/transposase system.

In the implementation scheme, the piggyBac method includes delivering to the TIL a (a) nucleic acid or amino acid sequence containing a sequence encoding a transposase and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon.

In one embodiment, the sequence encoding the transposase is an mRNA sequence. In another embodiment, the sequence encoding the transposase is a DNA sequence. In yet another embodiment, the DNA sequence is a cDNA sequence. In yet another embodiment, the sequence encoding the transposase is an amino acid sequence. The protein Superpiggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA.

Transposons/transposases

Exemplary transposon/transposase systems include, but are not limited to, piggyBac transposon and transposase, Sleeping Beauty transposon and transposase, Helraiser transposon and transposase, and Tol2 transposon and transposase.

The piggyBac transposon recognizes the transposon-specific inverted terminal repeat (ITR) at the end of the transposon and moves the contents between the ITRs to the TTAA chromosomal locus. The piggyBac transposon system has no effective load limitation on the gene of interest that can be included between the ITRs. In embodiments, the transposon is a piggyBac transposon or a piggyBac-like transposon.

Examples of piggyBac and piggyBac-like transposases and transposons include, for example, those disclosed in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is overactive. An overactive piggyBac or piggyBac-like transposase is a transposase that is more active than its naturally occurring variants. In embodiments, the overactive piggyBac or piggyBac-like transposase is isolated from or derived from the silkworm (Bombyxmori). A list of overactive amino acid substitutions is available in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is an integration-deficient type. In embodiments, an integration-deficient piggyBac or piggyBac-like transposase is a transposase capable of cleaving its corresponding transposon but integrating the cleaved transposon at a lower frequency than the corresponding wild-type transposase. A list of defective amino acid substitutions can be found in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

In the embodiments, the piggyBac or piggyBac-like transposon can be inserted into the 5'-TTAT-3 sequence within the target nucleic acid via a piggyBac or piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or SuperpiggyBac ^™ (SPB) transposase. In the embodiments, and particularly in embodiments where the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

The Sleeping Beauty (SB) transposon is translocated into the target genome via a Sleeping Beauty transposase that recognizes an ITR, and moves the contents between ITRs to the TA chromosomal locus. In an embodiment, the transposon is a Sleeping Beauty transposon. In an embodiment, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In an embodiment, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase.

The Helraiser transposon is translocated by the Helitron transposase. Unlike other transposases, the Helitron transposase does not contain an RNase-H-like catalytic domain, but instead contains a RepHel motif consisting of a replication initiation domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH nuclease superfamily. In embodiments, the transposon is the Helraiser transposon. In embodiments of the Helraiser transposon sequence, the transposase is flanked by left and right terminal sequences, referred to as the LTS and RTS. In embodiments, these sequences terminate with a conserved 5'-TC/CTAG-3' motif. In embodiments, a 19 bp palindromic sequence with the potential to form a hairpin termination structure is located 11 nucleotides upstream of the RTS and contains the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG. In embodiments, and particularly in embodiments where the transposon is the Helraiser transposon, the transposase is the Helitron transposase.

The Tol2 transposon can be isolated from or derived from the genome of the killifish and can be similar to transposons of the hAT family. An exemplary Tol2 transposon of this disclosure is encoded by a sequence comprising approximately 4.7 kilobases and contains a gene encoding a Tol2 transposase, said gene containing four exons. In embodiments, the transposon is a Tol2 transposon. In some embodiments of the methods of this disclosure, and particularly in those embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

In some embodiments, the vector comprises a recombinant and non-naturally occurring DNA sequence encoding a transposon. In some embodiments, the vector comprises DNA of any form, and wherein the vector comprises at least 100 nucleotides (nt), 500 nt, 1000 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6500 nt, 7000 nt, 7500 nt, 8000 nt, 8500 nt, 9000 nt, 9500 nt, 10,000 nt, or any number of nucleotides in between. In some embodiments, the vector comprises single-stranded or double-stranded DNA. In some embodiments, the vector comprises circular DNA. In some embodiments, the vector is a plasmid vector, a nanoplasmid vector, or a small loop. In some embodiments, the vector comprises linear or linearized DNA. In some embodiments, the vector is a double-stranded doggybone ^™ DNA sequence.

In the implementation scheme, the recombinant and non-naturally occurring DNA sequences encoding transposons also include sequences encoding one or more immune checkpoint genes.

In the implementation scheme, the nucleic acid sequence encoding the transposase is a DNA sequence, and the amount of the DNA sequence encoding the transposase and the DNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the DNA sequences encoding transposases and the DNA sequences encoding transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation scheme, the nucleic acid sequence encoding the transposase is an RNA sequence, and the amount of the RNA sequence encoding the transposase and the RNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the RNA sequences encoding transposases and transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation, the TIL is further modified by a second gene-editing tool, including but not limited to those described herein. In the implementation, the second gene-editing tool may include a piggyBac transposase that is only excised to re-excise the inserted sequence or any portion thereof. For example, a piggyBac transposase that is only excised can be used to "re-excise" a transposon.

According to some embodiments, the piggyBac system comprises a transposon/transposase system, wherein the transposase recognizes ITRs located flanking a transposon cassette containing a cargo encoding one or more immune checkpoint genes, and excises transposable elements to integrate it into a TT/AA chromosomal site, resulting in genomic insertion of the transposon cassette and expression of one or more immune checkpoint genes. According to some embodiments, the cargo encodes two or more immune checkpoint molecules.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the piggyBac method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD), and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using the piggyBac method and which can be used according to embodiments of the invention are described in WO2019/046815, WO2015006700, WO2010085699, WO2010099301, WO2010099296, WO2006122442, WO2001081565, and WO1998040510, the contents of which are incorporated herein by reference in their entirety.

Resources for executing the piggyBac method, such as plasmids for expressing transposons/transposases, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a piggyBac system, wherein the at least one gene editor system affects the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population and regulates the expression of at least one checkpoint protein in multiple cells of the second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) The second TIL population is obtained by stimulating it with OKT-3 and culturing it for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivery of at least one gene editor system comprising the piggyBac system, said gene editor system influencing the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population. In some embodiments, the at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

In some embodiments, methods for genetically modifying TIL populations include using non-viral techniques, such as the piggybac method (e.g., piggybac transposons and transposases or piggybac-like transposons and transposases). In some embodiments, the method includes delivering to the TIL: (a) a nucleic acid or amino acid sequence containing a sequence encoding a transposase; and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an mRNA sequence. The mRNA sequence encoding the transposase can be generated in vitro. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is a DNA sequence. The DNA sequence encoding the transposase can be generated in vitro. The DNA sequence may be a cDNA sequence. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an amino acid sequence. The amino acid sequence encoding the transposase can be generated in vitro. A protein super piggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA. In some embodiments, the transposon is a piggybac transposon or a piggybac-like transposon. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In some embodiments, and particularly those in which the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In some embodiments, the piggyBac transposase comprises the amino acid sequence containing SEQ ID NO:14487 of WO2019046815. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or Super piggyBac ^™ (SPB) transposase. In some embodiments, and particularly those in which the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence. In some embodiments of the methods disclosed herein, the transposase is a piggyBac ^™ (PB) transposase. piggyBac (PB) transposases may contain or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to the following sequences:

In some embodiments of the methods disclosed herein, the transposon is a Sleeping Beauty transposon. In some embodiments of the methods disclosed herein, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase. In some embodiments, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween the following sequences:

In some embodiments, including those in which the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof, identity with the following sequences:

V.Gen 2TIL Manufacturing Process

Figures 1 and 2 depict an exemplary TIL process family called Gen 2 (also known as process 2A) containing some of these features. An implementation of Gen 2 is shown in Figure 2.

As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs to enhance their metabolic activity and thus improve relative health status prior to transplantation into a patient, as well as methods for testing said metabolic health status. As generally outlined herein, TILs are typically obtained from patient samples and manipulated to amplify their number prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below.

In some implementations, TILs can be cryopreserved. After thawing, they can also be restimulated before being infused into the patient to increase their metabolism.

In some embodiments, the first amplification (including a process called pre-REP and the process shown as step B in Figures 1 and 36) is shortened to 3 to 14 days, and the second amplification (including a process called REP and the process shown as step D in Figure 1 or 36) is shortened to 7 to 14 days, as discussed in detail below, in the embodiments, and in the accompanying drawings. In some embodiments, the first amplification (e.g., the amplification described as step B in Figure 1 or 36) is shortened to 11 days, and the second amplification (e.g., the amplification described as step D in Figure 1 or 36) is shortened to 11 days. In some embodiments, the combination of the first and second amplifications (e.g., the amplification described as steps B and D in Figure 1 or 36) is shortened to 22 days, as discussed in detail below, in the embodiments, and in the accompanying drawings.

The "steps" denoted as A, B, C, etc. in the following text refer to Figure 1 or Figure 36 and certain embodiments described herein. The order of the steps in the following text and Figures 1 and 36 is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, repetitions of steps, and/or omissions of steps.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples and then expanded into larger populations for further manipulation as described herein, optionally cryopreservation, restimulation as outlined herein, and optionally evaluation of phenotypic and metabolic parameters as indicators of TIL health status.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multifocal sampling is used. In some embodiments, surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells include multifocal sampling (i.e., obtaining samples from one or more tumor sites and/or locations in a patient, as well as from one or more tumors at the same location or closely adjacent to each other). Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be lung tissue. In some embodiments, the applicable TIL is obtained from non-small cell lung cancer (NSCLC). Solid tumors can be skin tissue. In some embodiments, the applicable TIL is obtained from melanoma.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as HBSS.

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is a 10X working stock solution at a concentration of 100 mg/mL.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is a 10X working stock solution of 10,000 IU/mL.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is a 10X working stock solution at a concentration of 10 mg/mL.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatic tumor digests and tumor fragments obtained by digesting or fragmenting tumor samples obtained from patients.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as shown in Figure 1 or Figure 36), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor is 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor is 2mm × 2mm × 2mm. In some embodiments, the tumor is 3mm × 3mm × 3mm. In some embodiments, the tumor is 4mm × 4mm × 4mm.

In some embodiments, the tumor is removed to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of necrotic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of adipose tissue in each mass.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be used to remove these cells.

In some implementations, the cell suspension collected prior to the first amplification step is referred to as the “primary cell population” or “freshly collected” cell population.

In some embodiments, the cells may optionally be frozen after sample collection and cryopreserved before proceeding to the amplification described in step B (which is described in further detail below and in examples in Figures 1 and 36 and Figure 8).

1. Pleural effusion T cells and TIL

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may originate from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may originate from secondary metastatic cancer cells originating from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural transudate. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments where the disclosed methods utilize pleural fluid, ascites fluid or other cystic fluids containing TILs can be used to perform the same methods to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to further processing steps. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to further processing steps. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of viable TILs. If retained in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in further processing steps of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

B. Step B: First Amplification

In some embodiments, the method of the present invention achieves the acquisition of young TILs, which, compared to older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to the subject/patient), enable increased replication cycles at the time of administration to the subject/patient and thus provide additional therapeutic benefits. Characteristics of young TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32: 415-423; Robbins et al., J. Immunol. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, which are each incorporated herein by reference.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (connection region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 1 or Figure 36. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method referred to as Process 1C, as exemplified in Figures 5 and/or 6. In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased.

Following the dissection or digestion of the tumor fragment as described in step A of, for example, Figure 1 or Figure 36, the resulting cells are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 3 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 7 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for about 11 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells.

In some implementations, TIL amplification may be performed using the initial host TIL amplification steps described below and herein (e.g., the steps described in step B of Figure 1 or Figure 36, which may include a process called pre-REP), followed by a second amplification step described below and herein (step D, including a process called rapid amplification protocol (REP) step), followed by optional cryopreservation, and then a second step D described below and herein (including a process called restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic traits and metabolic parameters as described herein.

In TIL culture, the culture is initiated in a 24-well plate, such as using a Costar 24-well cell culture cluster, with a flat bottom (Corning Incorporated, Corning, NY). Each well is seeded with 2 mL of complete culture medium (CM) containing 1 × ^10⁶ tumor digested cells or one tumor fragment and IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digestion cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plate are incubated in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days.

After preparing the tumor fragment, the resulting cells (i.e., the fragment) are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum (or, in some cases, as outlined herein, in the presence of an APC cell population) and 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ primary TIL cells. In some embodiments, the growth medium contains IL-2 or a variant thereof during the initial expansion. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20-30 × ^10⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains IL-2 at concentrations between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL.

In some embodiments, the first amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the first amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the cell culture medium contains an anti-CD3 agonist antibody, such as OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, OKT-3 is present in the cell culture medium at the start of the first amplification (day 0). In some embodiments, OKT-3 is added to the cell culture medium at any time during the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 1 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 2 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 3 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 4 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 5 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 6 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 7 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 8 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 9 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 10 of the first amplification. In some implementations, OKT-3 is added to the cell culture medium on day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 1 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 3 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 6 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 7 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 8 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 9 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 10 and day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 2 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 3 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 4 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 5 of the first amplification.

In some implementations, OKT-3 is added once or more between day 5 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 6 of the first amplification.

In some implementations, OKT-3 is added once or more between day 6 and day 7 of the first amplification.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digest cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plates are cultured in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days. In some embodiments, CM is CM1 as described in the examples, see Example 1. In some embodiments, the first amplification is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 3-14 days, as discussed in the embodiments and figures. In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 7 to 14 days, as discussed in the embodiments and shown in FIG. 4 and FIG. 5, and includes amplification such as that described in step B of FIG. 1 or FIG. 36. In some embodiments, the first amplification in step B is shortened to 10-14 days. In some embodiments, the first amplification is shortened to 11 days, as discussed in the amplification described in step B of FIG. 1 or FIG. 36.

In some embodiments, the first TIL amplification can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the first TIL amplification can be performed for 1 to 14 days. In some embodiments, the first TIL amplification can be performed for 2 to 14 days. In some embodiments, the first TIL amplification can be performed for 3 to 14 days. In some embodiments, the first TIL amplification can be performed for 4 to 14 days. In some embodiments, the first TIL amplification can be performed for 5 to 14 days. In some embodiments, the first TIL amplification can be performed for 6 to 14 days. In some embodiments, the first TIL amplification can be performed for 7 to 14 days. In some embodiments, the first TIL amplification can be performed for 8 to 14 days. In some embodiments, the first TIL amplification can be performed for 9 to 14 days. In some embodiments, the first TIL amplification can be performed for 10 to 14 days. In some embodiments, the first TIL amplification can be performed for 11 to 14 days. In some embodiments, the first TIL amplification can proceed for 12 to 14 days. In some embodiments, the first TIL amplification can proceed for 13 to 14 days. In some embodiments, the first TIL amplification can proceed for 14 days. In some embodiments, the first TIL amplification can proceed for 1 to 11 days. In some embodiments, the first TIL amplification can proceed for 2 to 11 days. In some embodiments, the first TIL amplification can proceed for 3 to 11 days. In some embodiments, the first TIL amplification can proceed for 4 to 11 days. In some embodiments, the first TIL amplification can proceed for 5 to 11 days. In some embodiments, the first TIL amplification can proceed for 6 to 11 days. In some embodiments, the first TIL amplification can proceed for 7 to 11 days. In some embodiments, the first TIL amplification can proceed for 8 to 11 days. In some embodiments, the first TIL amplification can proceed for 9 to 11 days. In some embodiments, the first TIL amplification can proceed for 10 to 11 days. In some embodiments, the first TIL amplification can proceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the first amplification period. In some embodiments, the first amplification period, including, for example, during step B of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, as discussed in the examples and figures, the first amplification process (including a process called pre-REP; for example, step B according to Figure 1 or Figure 36) is shortened to 3 to 14 days. In some embodiments, the first amplification in step B is shortened to 7 to 14 days. In some embodiments, the first amplification in step B is shortened to 10 to 14 days. In some embodiments, the first amplification is shortened to 11 days.

In some embodiments, the first amplification is performed in a closed-system bioreactor, such as step B according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

1. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Transition from the first amplification to the second amplification

In some cases, the host TIL population obtained from the first amplification, including, for example, the TIL population obtained from step B as shown in Figure 1 or Figure 36, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first amplification (referred to as the second TIL population) can undergo a second amplification (which may include an amplification sometimes referred to as REP) and then be cryopreserved as discussed below. Similarly, where the genetically modified TILs are intended for therapeutic use, the first TIL population (sometimes referred to as the host TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be genetically modified before amplification or after the first amplification and before the second amplification for use in a suitable treatment.

In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is stored until a phenotypic analysis for selection is performed. In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is not stored and proceeds directly to the second amplification. In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 10 days after fragmentation. In some implementations, the transition from the first amplification to the second amplification occurs approximately 7 to 14 days after fragmentation. In other implementations, the transition from the first amplification to the second amplification occurs approximately 14 days after fragmentation.

In some embodiments, the transition from the first amplification to the second amplification occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 1 to 14 days after fragmentation. In some embodiments, the first TIL amplification may occur 2 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 5 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 7 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after fragmentation. In some embodiments, the transition occurs 9 to 14 days after fragmentation. In some embodiments, the transition occurs 10 to 14 days after fragmentation. In some embodiments, the transition occurs 11 to 14 days after fragmentation. In some embodiments, the transition occurs 12 to 14 days after fragmentation. In some embodiments, the transition occurs 13 to 14 days after fragmentation. In some embodiments, the transition occurs 14 days after fragmentation. In some embodiments, the transition occurs 1 to 11 days after fragmentation. In some embodiments, the transition occurs 2 to 11 days after fragmentation. In some embodiments, the transition occurs 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 11 days after fragmentation. In some embodiments, the transition occurs 5 to 11 days after fragmentation. In some embodiments, the transition occurs 6 to 11 days after fragmentation. In some embodiments, the transition occurs 7 to 11 days after fragmentation. In some embodiments, the transition occurs 8 to 11 days after fragmentation. In some embodiments, the transition occurs 9 to 11 days after fragmentation. In some embodiments, the transition occurs 10 to 11 days after fragmentation. In some embodiments, the transition occurs 11 days after fragmentation.

In some embodiments, TILs are not stored after the first amplification and before the rapid second amplification, and the TILs undergo the second amplification directly (e.g., in some embodiments, no storage occurs during the transition from steps B to D as shown in Figure 1 or Figure 36). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from the first amplification (the second TIL population) undergo the second amplification directly without a transition period.

In some embodiments, the transition from the first amplification to the second amplification (e.g., step C according to Figure 1 or Figure 36) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

According to this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from MiltenyiBiotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulation of TIGIT expression and downregulation of one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step also includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Second amplification

In some embodiments, the number of TIL cell populations is expanded after collection and initial host treatment, for example, after steps A and B and a transition referred to as step C (as indicated in Figure 1 or Figure 36). This further expansion is referred to herein as a second expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP); and the process indicated in step D of Figure 1 or Figure 36. The second expansion is typically performed in a breathable container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 1 or Figure 36) may be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL amplification may be performed for about 7 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 8 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 9 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 10 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 11 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 12 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 13 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 14 days.

In some embodiments, the second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the procedures indicated in step D of Figure 1 or Figure 36). For example, TILs can be rapidly amplified using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as about 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (available from BioLegend, San Diego, CA). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation to the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of: urinumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, the second amplification period, including, for example, during step D of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step D of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, during rapid expansion and/or secondary expansion, the ratio of TILs to PBMCs and/or antigen-presenting cells is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:100 and 1:200.

In some implementations, REP and/or secondary amplification are performed in culture flasks, where the host TIL is mixed with 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. The medium is replaced (typically 2/3 replaced with fresh medium via aspiration) until the cells are transferred to a replacement growth chamber. The replacement growth chamber comprises G-REX culture flasks and aeration containers, as discussed more fully below.

In some embodiments, the second amplification (which may include a process referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second amplification is shortened to 11 days.

In some embodiments, the REP and/or second amplification can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or aeration dishes (G-REX culture flasks). In some embodiments, the second amplification (including amplification referred to as rapid amplification) is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs suspended in 150 mL of medium can be added to each T-175 culture flask. The TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The T-175 culture flasks can be incubated at 37°C and 5% _CO₂ . Half of the medium can be replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some implementations, on day 7, cells from two T-175 culture flasks can be combined in a 3L bag, and 300 mL of AIM V with 5% human AB serum and 3000 IU/mL IL-2 can be added to 300 mL of TIL suspension. The cell count in each bag is counted daily or every two days, and fresh culture medium is added to maintain the cell count between 0.5 and 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (which may include an amplification referred to as REP, as well as the amplification mentioned in step D of Figure 1 or Figure 36) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing, New Brighton, MN, USA) with a 100 cm aerated silica bottom. 5 × ^10⁶ or 10 × ^10⁶ TILs can be cultured together with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). The G-REX-100 culture flask can be incubated at 37°C in 5% _CO₂ . On day 5, 250 mL of the supernatant can be removed and transferred to a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. TIL precipitate can be resuspended in 150 mL of fresh culture medium containing 5% human AB serum and 3000 IU/mL IL-2, and added back to the initial G-REX-100 culture flask. When TILs are serially amplified in G-REX-100 culture flasks, on day 7, TILs from each G-REX-100 flask can be resuspended in 300 mL of culture medium present in each flask, and the cell suspension can be aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 can be added to each culture flask. The G-REX-100 culture flasks can be incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 can be added to each G-REX-100 culture flask. Cells can be harvested on day 14 of culture. In some implementations, the process uses different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different number of repetitions.

In some embodiments, the second amplification (including an amplification termed REP) is performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some embodiments, two-thirds of the medium is replaced with fresh medium by aspiration. In some embodiments, the alternative growth chamber comprises a G-REX culture flask and a ventilated container, as discussed more fully below.

In some embodiments, a second amplification (including an amplification referred to as REP) is performed, and the process also includes a step of selecting the TIL for superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability assays may be performed after a second amplification (including amplification known as REP amplification) using standard assays known in the art. For example, a trypan blue exclusion assay may be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample may be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, the second amplification of TILs (including amplification referred to as REP) can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., 2008, Jimmunother., 31, 742-751, and Dudley et al., 2003, Jimmunother., 26, 332-342) or aeration G-REX culture flasks. In some embodiments, the second amplification is performed using culture flasks. In some embodiments, the second amplification is performed using aeration G-REX culture flasks. In some embodiments, the second amplification is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs are suspended in approximately 150 mL of culture medium and added to each T-175 culture flask. TILs were cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs used as "feeder" cells, and the cells were cultured in a 1:1 mixture (50/50 medium) of CM and AIM-V supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. T-175 culture flasks were incubated at 37°C and 5% _CO2 . In some embodiments, half of the medium was replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 culture flasks were pooled in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 was added to 300 mL of TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh culture medium can be added to maintain the cell count between approximately 0.5 and approximately 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (including an amplification referred to as REP) is performed in a 500 mL volumetric flask (G-REX-100, Wilson Wolf) with a 100 ^cm² permeable silica bottom. Approximately 5 × ^10⁶ or 10 × ^10⁶ TILs are incubated with irradiated allogeneic PBMCs at a 1:100 ratio in 400 mL of 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The G-REX-100 flask is incubated at 37°C and 5% _CO₂ . In some embodiments, on day 5, 250 mL of the supernatant is removed and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL precipitate is then resuspended in 150 mL of fresh 50/50 medium with 3000 IU/mL IL-2 and added back to the initial G-REX-100 flask. In the embodiment of continuous TIL amplification in G-REX-100 culture flasks, on day 7, the TIL from each G-REX-100 flask is resuspended in 300 mL of culture medium present in each flask, and the cell suspension is aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 is added to each culture flask. The G-REX-100 culture flasks are incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 is added to each G-REX-100 culture flask. Cells are harvested on day 14 of culture. In some embodiments, the process is performed using different centrifugation speeds (400 g, 300 g, 200 g, for 5 minutes) and different number of repetitions.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some implementations, the second amplification medium (e.g., sometimes referred to as CM2 or second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the second amplification is performed in a closed-system bioreactor, such as step D according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve vertical scaling up of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve lateral scaling out of the culture by: (a) performing rapid or second amplification by culturing TILs in a first small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the TILs transferred from the first small-scale culture is cultured in a second small-scale culture for approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 5 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid or second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid or second amplification is performed for a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid or second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid or second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid or secondary amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid or secondary amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains the same components as the cell culture medium used for rapid or secondary expansion after splitting. In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains different components than the cell culture medium used for rapid or secondary expansion after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid or secondary expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

1. Feeder cells and antigen-presenting cells

In some embodiments, the second amplification procedure described herein (e.g., including the amplification described in step D of Figure 1 or Figure 36, and an amplification referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells placed into the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some implementations, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 50 × ^10⁶ TILs. In still other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 25 × ^10⁶ TILs.

In some embodiments, the second amplification procedure described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs in the second expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796A1, the disclosure of which is incorporated herein by reference.

Step E: Collect TIL

Cells can be collected after the second amplification step. In some embodiments, TILs are collected after one, two, three, four, or more amplification steps, as shown in, for example, Figure 1 or Figure 36. In some embodiments, TILs are collected after two amplification steps, as shown in, for example, Figure 1 or Figure 36.

TILs can be collected by any suitable and aseptic method, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the methods of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, collection, such as step E according to Figure 1 or Figure 36, is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, step E according to Figure 1 or Figure 36 is performed according to the process described herein. In some embodiments, the closed system is introduced under aseptic conditions via a syringe to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is employed.

In some embodiments, TIL is collected according to the methods described in the embodiments. In some embodiments, TIL between day 1 and day 11 is collected using the methods described in the steps mentioned herein, such as the day 11 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 24 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 22 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments.

Step F: Final preparation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 1 or Figure 36 and outlined in detail above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, TILs amplified using the APCs of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using the PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VI. Gen 3TIL Manufacturing Process

Without being limited by any particular theory, it is believed that the initiation of a first expansion to initiate T cell activation, followed by a rapid second expansion to enhance T cell activation, as described in the method of the present invention, allows for the preparation of expanded T cells that retain a "younger" phenotype, and therefore it is anticipated that the expanded T cells of the present invention will exhibit higher cytotoxicity against cancer cells compared to T cells expanded by other methods. In particular, it is believed that initiating T cell activation by exposure to anti-CD3 antibodies (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), as taught in the method of the present invention, and then enhancing it by subsequent exposure to additional anti-CD3 antibodies (e.g., OKT-3), IL-2, and APCs, limits or avoids the maturation of T cells in the culture medium, thereby producing a population of T cells with a less mature phenotype, which are less exhausted by culture expansion and exhibit higher cytotoxicity against cancer cells. In some implementations, the rapid second expansion step is broken down into multiple steps to achieve vertical scaling up of culture scale by: (a) performing rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the T cells from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container and culturing the T cells from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the T cells transferred from the first small-scale culture is cultured in the second small-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs. In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some implementations, after rapid expansion, multiple subpopulations contain a therapeutically effective amount of TIL. In some implementations, after rapid expansion, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some implementations, after rapid expansion, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, rapid amplification is performed over a period of approximately 1 to 5 days before being split into multiple steps. In some implementations, the splitting of rapid amplification occurs approximately day 1, day 2, day 3, day 4, or day 5 after the start of rapid amplification.

In some implementations, the split of rapid amplification occurs approximately day 8, 9, 10, 11, 12, or 13 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of rapid amplification occurs approximately day 10 after the start of the first amplification. In another exemplary implementation, the split of rapid amplification occurs approximately day 11 after the start of the first amplification.

In some implementations, rapid amplification is performed for a period of approximately 4 to 11 days after the split. In some implementations, rapid amplification is performed for a period of approximately 3, 4, 5, 6, 7, 8, 9, 10, or 11 days after the split.

In some embodiments, the cell culture medium used for rapid expansion before splitting contains the same components as the cell culture medium used for rapid expansion after splitting. In some embodiments, the cell culture medium used for rapid expansion before splitting contains different components than the cell culture medium used for rapid expansion after splitting.

In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, the fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

In some implementations, rapid second expansion is performed after the T cell activation achieved by initiating the first expansion begins to decrease, slow down, decline, or subside.

In some cases, rapid second expansion occurs when T cell activation, achieved through initiating the first expansion, has decreased by just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, or 47%. The percentages are 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 100%.

In some implementations, the rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

In some implementations, rapid second expansion occurs when T cell activation achieved by initiating the first expansion has decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46%. The percentages are 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after that.

In some cases, rapid second expansion occurs when T cell activation achieved through initiating first expansion has decreased to at most just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%. %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, the reduction in T cell activation achieved by initiating the first expansion is determined by a decrease in the amount of interferon-γ released by T cells in response to antigen stimulation.

In some implementations, the initial expansion of T cells occurs within a period of at most exactly 7 days or about 8 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or approximately 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, the initial expansion of T cells is carried out over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, rapid secondary expansion of T cells takes place over a period of at most exactly or about 11 days.

In some implementations, rapid secondary expansion of T cells is carried out within a period of at most exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, rapid secondary expansion of T cells is carried out over time periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 11 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 8 days, and the rapid second expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 9 days.

In some implementations, the initial expansion of T cells occurs over an 8-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 9 days.

In some implementations, the initial expansion of T cells occurs over a 7-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, T cells are tumor-infiltrating lymphocytes (TILs).

In some implementations, the T cells are bone marrow infiltrating lymphocytes (MIL).

In some implementations, the T cells are peripheral blood lymphocytes (PBLs).

In some implementations, the T cells are obtained from a donor with cancer.

In some implementations, T cells are TILs obtained from tumors removed from patients with cancer.

In some implementations, the T cells are MILs obtained from the bone marrow of patients with hematological malignancies.

In some implementations, the T cells are peripheral blood mononuclear cells (PBMCs) derived from a donor. In some implementations, the donor has cancer. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the donor has a tumor. In some implementations, the tumor is a liquid tumor. In some implementations, the tumor is a solid tumor. In some implementations, the donor has a hematological malignancy.

In some aspects of this disclosure, immune effector cells (e.g., T cells) can be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL separation). In a preferred aspect, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected via apheresis may be washed to remove plasma fractions and optionally placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations. In one aspect, T cells are separated from peripheral blood lymphocytes by lysing erythrocytes and, for example, by depleting monocytes through PERCOLL gradient centrifugation or by countercurrent centrifugation.

In some embodiments, the T cells are PBLs isolated from whole blood or lymphocyte-rich apheresis products from a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematologic malignancy. In some embodiments, PBL is isolated from whole blood or lymphocyte-rich apheresis products using a positive or negative selection method, i.e., removing PBL using markers of the T-cell phenotype (e.g., CD3+CD45+), or removing non-T-cell phenotype cells while leaving PBL. In other embodiments, PBL is separated by gradient centrifugation. After PBL is isolated from donor tissue, the initial amplification of PBL can be initiated according to the initial amplification step of any method described herein by seeding a suitable number of isolated PBLs (approximately 1 × ^10⁷ PBLs in some embodiments) into an initial amplification culture.

An exemplary TIL process containing some of these features, referred to as process 3 (also referred to herein as Gen 3), is depicted in FIG8 (in particular, for example, FIG8B and/or FIG8C and/or FIG8D) and FIG36, and some advantages of this embodiment of the invention compared to Gen 2 are described in FIG1, FIG2, FIG8, FIG30 and FIG31 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). Implementations of Gen 3 are shown in Figures 1, 8, 30, and 36 (particularly, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). Process 2A or Gen 2 is also described in U.S. Patent Publication No. 2018/0280436, which is incorporated herein by reference in its entirety. The Gen 3 process is also described in International Patent Publication WO 2020/096988.

As discussed and generally summarized herein, TILs are derived from patient samples and manipulated using a TIL amplification process described herein and referred to as Gen 3 to amplify their number before transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below. In some embodiments, TILs may be cryopreserved before or after amplification. After thawing, they may also be restimulated to increase their metabolism before infusion into a patient.

In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the process referred to herein as the Rapid Amplification Protocol (REP) and the process shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 7 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (in particular, e.g., Figure 8B and/or Figure 8C)) takes 1 to 7 days, and the rapid second amplification (including the process referred herein as rapid amplification protocol (REP), and the process shown as step D in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) takes 1 to 10 days, as discussed in detail below and in the embodiments and figures. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 9 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some embodiments, the combination of initiating the first amplification and rapid second amplification (e.g., amplification described in Figure 8 (particularly, e.g., Figure 8B and/or Figure 8C as steps B and D) lasts 14-16 days, as discussed in detail below and in the examples and figures. In particular, it is believed that certain embodiments of the invention include an initiating first amplification step, wherein TILs are activated by exposure to an anti-CD3 antibody (e.g., OKT-3) in the presence of IL-2 or by exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody (e.g., OKT-3). In some embodiments, the TILs activated in the initiating first amplification step as described above are a first TIL population, i.e., a primary cell population.

The “step” designations A, B, C, etc. in the following text are non-limiting examples with reference to Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and certain non-limiting embodiments described herein. The order of steps in the following text and Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, step repetitions, and/or step omissions.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples (“primary TILs”) or from circulating lymphocytes (e.g., peripheral blood lymphocytes, including peripheral blood lymphocytes with TIL-like characteristics), and are then expanded into larger populations for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotype and metabolic parameters as indicators of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be any type of cancer, including (but not limited to) breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including (but not limited to) squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the applicable TIL is obtained from malignant melanoma tumors because reports indicate that these tumors have particularly high levels of TIL.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. TILs are cultured from these fragments using enzymatically digested tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociating enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a certain amount of sterile buffer (e.g., HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is 100 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally, cell suspensions obtained from tumors are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as provided in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), the tumor is physically fragmented. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor fragments are 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor fragments are 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2mm × 2mm × 2mm. In some embodiments, the tumor fragment is 3mm × 3mm × 3mm. In some embodiments, the tumor fragment is 4mm × 4mm × 4mm.

In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue in each fragment. In some embodiments, the tumor fragmentation step is an in vitro or ex vivo method.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, the cell suspension prior to initiating the first amplification step is referred to as the “primary cell population” or “freshly acquired” or “freshly isolated” cell population.

In some embodiments, the cells may optionally be frozen after sample separation (e.g., after obtaining a tumor sample and/or after obtaining a cell suspension from a tumor sample) and cryopreserved before proceeding to the amplification described in step B, which is further described in detail below and illustrated in Figure 8 (particularly, for example, Figure 8B).

1. TIL derived from hollow needles/small biopsies

In some implementations, TILs are initially obtained from patient tumor samples (“primary TILs”) obtained through core needle biopsy or similar procedures and then expanded into a larger population for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters.

In some embodiments, patient tumor samples may be obtained using methods known in the art, typically via mini-biopsy, core needle biopsy, needle biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples may be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples may also be liquid tumors, such as those obtained from hematologic malignancies. In some embodiments, the sample may be derived from multiple small tumor samples or biopsies. In some embodiments, the sample may comprise multiple tumor samples from a single tumor from the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three, or four tumors from the same patient. In some embodiments, the sample may comprise multiple tumor samples from multiple tumors from the same patient. The solid tumor may be lung and/or non-small cell lung cancer (NSCLC).

Generally, cell suspensions obtained from the tumor core or fragments are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

In some implementations, if the tumor is metastatic and the primary lesion has been effectively treated/removed in the past, it may be necessary to remove a metastatic lesion. In some implementations, when available, minimally invasive methods are used to remove skin lesions or lymph nodes in the neck or axillary region. In some implementations, skin lesions are removed or small biopsies are taken. In some implementations, lymph nodes are removed or small biopsies are taken. In some implementations, the tumor is melanoma. In some implementations, the small biopsy of melanoma includes a mole or a portion thereof.

In some embodiments, a small biopsy is a perforated biopsy. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin surrounding a suspected mole. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin and removing a circular piece of skin. In some embodiments, a small biopsy is a perforated biopsy and removes a circular portion of the tumor.

In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed along with a small border of normal-looking skin.

In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy and only the most irregular portion of the mole or growth is collected. In some implementations, the small biopsy is an incisional biopsy, and the incisional biopsy is used when other techniques cannot be performed, such as when the suspected mole is very large.

In some implementations, the small biopsy is a lung biopsy. In some implementations, the small biopsy is obtained via bronchoscopy. Typically, bronchoscopy involves inserting a small instrument through the nose or mouth, down the throat, and into the bronchial passage under the patient's anesthesia, where the instrument is used to remove tissue. In some implementations, a transthoracic biopsy may be used when the tumor or growth cannot be reached via bronchoscopy. Typically, for a transthoracic biopsy, the patient is also under anesthesia and a needle is inserted directly through the skin to the suspected site to remove a small tissue sample. In some implementations, a transthoracic biopsy may require interventional radiology (e.g., using X-ray or CT scan-guided needle). In some implementations, the small biopsy is obtained via puncture biopsy. In some implementations, the small biopsy is obtained via endoscopic ultrasound (e.g., an endoscope with a light and placed in the esophagus via the mouth). In some implementations, the small biopsy is obtained surgically.

In some implementations, the small biopsy is a head and neck biopsy. In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy in which a small piece of tissue is removed from the area of abnormal appearance. In some implementations, if the abnormal area is easily accessible, a sample can be collected without hospitalization. In some implementations, if the tumor is deep inside the mouth or throat, the biopsy may need to be performed under general anesthesia in an operating room. In some implementations, the small biopsy is an excisional biopsy. In some implementations, the small biopsy is an excisional biopsy in which the entire area is removed. In some implementations, the small biopsy is a fine-needle aspiration (FNA). In some implementations, the small biopsy is a fine-needle aspiration (FNA) in which cells are extracted (aspirated) from the tumor or mass using a very fine needle attached to a syringe. In some implementations, the small biopsy is a perforated biopsy. In some implementations, the small biopsy is a perforated biopsy in which a suspicious area is removed using perforated forceps.

In some implementations, the small biopsy is a cervical biopsy. In some implementations, the small biopsy is obtained via colposcopy. Typically, the colposcopy method uses a light-assisted magnifying instrument (coloscope) attached to a binocular magnifying lens, which is then used to examine a small portion of the cervix. In some implementations, the small biopsy is a cervical conization/cone biopsy. In some implementations, a small biopsy is a cervical conization/cone biopsy, in which an outpatient procedure may be required to remove a larger piece of tissue from the cervix. In some implementations, in addition to aiding in diagnosis, a cone biopsy can also be used as initial treatment.

The term "solid tumor" refers to an abnormal mass of tissue that typically does not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The histological structure of a solid tumor includes interdependent tissue compartments, comprising the parenchyma (cancer cells) and supporting stromal cells dispersed within it, providing a supportive microenvironment.

In some embodiments, samples from the tumor are obtained in the form of fine-needle aspiration (FNA), core needle biopsy, or mini-biopsy (including, for example, perforated biopsy). In some embodiments, the sample is first placed in a G-REX-10. In some embodiments, when one or two core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-10. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-100. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-500.

FNA can be obtained from skin tumors, including, for example, melanoma. In some implementations, the FNA is obtained from a skin tumor, such as one from a patient with metastatic melanoma. In some cases, the patient with melanoma has previously undergone surgical treatment.

FNA can be derived from lung tumors, including, for example, NSCLC. In some implementations, the FNA is derived from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the NSCLC patient has previously undergone surgical treatment.

The TILs described herein may be obtained from FNA samples. In some cases, FNA samples are obtained or separated from the patient using fine-gauge needles ranging from 18 to 25 gauge. Fine-gauge needles may be 18, 19, 20, 21, 22, 23, 24, or 25 gauge. In some embodiments, the FNA sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

In some cases, the TILs described herein are obtained from core needle biopsy samples. In some cases, the core needle biopsy sample is obtained or separated from the patient using a surgical or medical needle in the range of 11 to 16 gauge. The needle may be 11, 12, 13, 14, 15, or 16 gauge. In some embodiments, the core needle biopsy sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some implementations, the TIL is not derived from tumor digest. In some implementations, the solid tumor core is not fragmented.

In some embodiments, the tumor digest is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically dissociated again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically dissociated a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether it is incubated at 37°C in 5% _CO2 for another 30 minutes. In some implementations, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, obtaining the first TIL population involves a multifocal sampling method.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as Hank's balance salt solution (HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some implementations, the enzyme stock solution may be varied, thus requiring verification of the concentration of the lyophilized stock solution and corresponding modification of the final amount of enzyme added to the digestion mixture.

In some embodiments, the enzyme mixture comprises approximately 10.2 μl of neutral protease (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

2. Pleural effusion T cells and TILs

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may be derived from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells derived from another organ (e.g., breast, ovary, colon, or prostate). In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural effusion. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments of the pleural fluid in the examples of the present invention, ascites fluid or other cyst fluid containing TILs can be used for the same method to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to the contact step. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to the contact step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of live TILs. If retained in untreated pleural fluid, the number of live TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in the contact step is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in the contact step of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

3. Methods for expanding peripheral blood lymphocytes (PBL) from peripheral blood.

PBL Method 1. In some embodiments of the invention, PBL is amplification using the methods described herein. In some embodiments of the invention, the method includes obtaining a PBMC sample from whole blood. In some embodiments, the method includes enriching T cells by using negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC. In some embodiments, the method includes enriching T cells by using magnetic bead-based negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC.

In some embodiments of the present invention, PBL method 1 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. T cells are isolated using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

PBL Method 2. In some embodiments of the invention, PBL is amplified using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells from the PBMCs are enriched by incubating the PBMCs at 37°C for at least three hours and then separating non-adhesive cells.

In some embodiments of the present invention, PBL method 2 is performed as follows: On day 0, the frozen PMBC sample is thawed, and PBMC cells are seeded at 6 million cells per well in 6-well plates of CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, the non-adherent cells (which are PBL) are removed and their number is counted.

PBL Method 3. In some embodiments of the invention, PBL is amplified using PBL Method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection and T cells are selected using a non-CD19+ fraction of the PBMC sample using negative selection.

In some embodiments of the present invention, PBL method 3 is performed as follows: On day 0, frozen PBMCs derived from peripheral blood are thawed and their number is counted. CD19+ B cells are sorted using a CD19 multi-sorting human kit (Miltenyi Biotec). In the non-CD19+ cell fraction, T cells are purified using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

In some embodiments, PBMCs are isolated from whole blood samples. In some embodiments, PBMC samples are used as starting material for amplifying PBL. In some embodiments, the samples are cryopreserved prior to the amplification process. In other embodiments, fresh samples are used as starting material for amplifying PBL. In some embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human pan-T cell isolation kit and an LS column. In some embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art (e.g., CD19 negative selection).

In some embodiments of the invention, the PBMC sample is incubated for a period of time at a temperature required for effective identification of non-adhesive cells. In some embodiments of the invention, the incubation time is approximately 3 hours. In some embodiments of the invention, the temperature is approximately 37°C. The non-adhesive cells are then amplified using the process described above.

In some embodiments, PBMC samples are from subjects or patients who have optionally pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, tumor samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, PBMC samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or longer. In other embodiments, PBMCs are from patients currently receiving an ITK inhibitor regimen (e.g., ibrutinib).

In some implementations, PBMC samples are derived from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor and who are difficult to treat with a kinase inhibitor or an ITK inhibitor (e.g., ibrutinib).

In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment. In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment and have not been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or longer. In other embodiments, PBMCs are derived from patients who have previously been exposed to ITK inhibitors but have not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In some embodiments of the invention, on day 0, cells are selected for CD19+ and sorted accordingly. In some embodiments of the invention, antibody-bound beads are used for selection. In some embodiments of the invention, purified T cells are isolated from PBMCs on day 0.

In some embodiments of the present invention, for patients who have not been pretreated with ibrutinib or other ITK inhibitors, 10-15 mL of erythrocyte sedimentation rate (ESR) amber layer will produce approximately 5 × ^10⁹ PBMCs, which in turn will produce approximately 5.5 × ^10⁷ PBLs.

In some embodiments of the invention, for patients pretreated with ibrutinib or other ITK inhibitors, the amplification process will generate approximately 20 × ^10⁹ PBLs. In some embodiments of the invention, 40.3 × ^10⁶ PBMCs will generate approximately 4.7 × ^10⁵ PBLs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or obtained from any other method known in the art for obtaining PBMCs.

In some embodiments, the PBL is prepared using the method described in U.S. Patent Application Publication No. US 2020/0347350A1, the disclosure of which is incorporated herein by reference.

4. Methods for expanding bone marrow infiltrating lymphocytes (MILs) derived from bone marrow-derived PBMCs

MIL Method 3. In some embodiments of the invention, the method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+/CD33+/CD20+/CD14+ cells, and non-CD3+/CD33+/CD20+/CD14+ cell fractions are sonicated and a portion of the sonicated cell fractions are added back to the selected cell fractions.

In some embodiments of the present invention, MIL method 3 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. Cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using an S3e cell sorter (Bio-Rad). Cells are sorted into two fractions: immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and AML embryonic cell fraction (non-CD3+CD33+CD20+CD14+).

In some embodiments of the invention, PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow via apheresis, aspiration, biopsy, or other similar methods known in the art. In some embodiments, PBMCs are fresh. In other embodiments, PBMCs are cryopreserved.

In some embodiments of the invention, the MIL is expanded from 10-50 mL of bone marrow aspirate. In some embodiments of the invention, 10 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 20 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 30 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 40 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 50 mL of bone marrow aspirate is obtained from the patient.

In some embodiments of the invention, the number of PBMCs produced from about 10-50 mL of bone marrow aspirate is about 5 × ^10⁷ to about 10 × ^10⁷ PBMCs. In other embodiments, the number of PBMCs produced is about 7 × ^10⁷ PBMCs.

In some embodiments of the invention, approximately 5 × ^10⁷ to approximately 10 × ^10⁷ PBMCs produce approximately 0.5 × ^10⁶ to approximately 1.5 × ^10⁶ MILs. In some embodiments of the invention, approximately 1 × ^10⁶ MILs are produced.

In some embodiments of the invention, 12 × ^10⁶ PBMCs derived from bone marrow aspirate yield approximately 1.4 × ^10⁵ MILs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, bone marrow, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or from any other method known in the art for obtaining PBMCs.

In some embodiments, the MIL is prepared using the method described in U.S. Patent Application Publication No. US2020/0347350A1, the disclosure of which is incorporated herein by reference.

Step B: Initiating the first amplification

In some embodiments, the method of the present invention provides younger TILs that may offer additional therapeutic benefits compared to older TILs (i.e., TILs that have undergone further replication in multiple rounds prior to administration to the subject/patient). Characteristics of younger TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32, 415-423; Robbins et al., J. Immunother. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, each of which is incorporated herein by reference.

Following the segmentation or digestion of tumor fragments and/or tumor fragments as described in step A of, for example, Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) under conditions favorable to TIL growth but unfavorable to tumor and other cell growth. In some embodiments, IL-2, OKT-3, and feeder cells are added together with the tumor digest and/or tumor fragments at the start of culture (e.g., on day 0). In some embodiments, the tumor digest and/or tumor fragments are incubated in containers at a concentration of up to 60 fragments per container and with 6000 IU/mL IL-2. In some embodiments, culturing this primary cell population for several days, typically 1 to 8 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, culturing this primary cell population for several days, typically 1 to 7 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells.

In some implementations, TIL amplification may be performed using the steps described in step B of Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), which may include a process called pre-REP or initiating REP and which begins on day 0 and/or from the start of culture containing feeder cells), followed by a rapid second amplification (step D, including a process called the Rapid Amplification Protocol (REP) step) as described in step D below and here, followed by optional cryopreservation, and then a second step D as described below and here (including a process called the restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some implementations, the tumor fragments are between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.

In some embodiments, there are fewer or equal to 240 tumor fragments. In some embodiments, fewer or equal to 240 tumor fragments are placed in fewer or equal to 4 containers. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, fewer or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each container contains fewer or equal to 500 mL of culture medium. In some embodiments, the culture medium contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the culture medium contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains OKT-3. In some embodiments, the culture medium contains 30 ng/mL OKT-3 per container. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells ^per container.

After preparing the tumor fragment, the resulting cells (i.e., fragments of the primary cell population) are cultured in a medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions favorable to TIL growth but unfavorable to tumor and other cell growth, allowing TIL initiation and accelerated growth from day 0. In some embodiments, the tumor digest and/or tumor fragment are incubated with 6000 IU/mL IL-2, antigen-presenting feeder cells, and OKT-3. This primary cell population is cultured for several days, typically 1 to 8 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initial expansion period contains IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for several days, typically 1 to 7 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initiation of the first expansion comprises IL-2 or a variant thereof, along with antigen-presenting feeder cells and OKT-3. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20-30 ^{× 10⁶} IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × 10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the initial amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some embodiments, the initial expansion cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or about 8000 IU/mL IL-2.

In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial expansion medium contains approximately 200 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium also contains IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 2 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the initial cell culture medium for expansion contains OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, the initial amplification cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 6000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the initiation first expansion medium is referred to as "CM" (an abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In some embodiments, CM is CM1 as described in the examples. In some embodiments, the initiation of the first expansion is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initiation first expansion medium or initial cell culture medium or first cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (also referred to herein as feeder cells).

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific). In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some implementations, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisherScientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the specific culture medium described by reference to International PCT Publication WO/1998/030679, which is incorporated herein by reference, can be used in this invention. In that disclosure, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4+, Br, T, ^{Mn2 +} , P, Si4+, V5 ⁺ , Mo6 ⁺ , ^{Ni2 +} , Rb+, Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

Table 4: Concentration of Non-Trace Element Components

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) can be used in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 4 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 5 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG1 or FIG36 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 7 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 4 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8B and/or FIG8C), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 5 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 7 days, as discussed in the embodiments and figures.

In some embodiments, the initiation of first TIL amplification may be performed 1 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 1 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 7 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some implementations, the initiation of the first TIL amplification can be performed 7 days after fragmentation occurs and/or after the start of the first initiation amplification step.

In some implementations, the initial amplification of the TIL can take place for 1, 2, 3, 4, 5, 6, 7, or 8 days. In some implementations, the first TIL amplification can take place for 1 to 8 days. In some implementations, the first TIL amplification can take place for 1 to 7 days. In some implementations, the first TIL amplification can take place for 2 to 8 days. In some implementations, the first TIL amplification can take place for 2 to 7 days. In some implementations, the first TIL amplification can take place for 3 to 8 days. In some implementations, the first TIL amplification can take place for 3 to 7 days. In some implementations, the first TIL amplification can take place for 4 to 8 days. In some implementations, the first TIL amplification can take place for 4 to 7 days. In some implementations, the first TIL amplification can take place for 5 to 8 days. In some implementations, the first TIL amplification can take place for 5 to 7 days. In some implementations, the first TIL amplification can take place for 6 to 8 days. In some implementations, the first TIL amplification can take place for 6 to 7 days. In some implementations, the first TIL amplification can take place for 7 to 8 days. In some implementations, the first TIL amplification can take 8 days. In some implementations, the first TIL amplification can take 7 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, during the initiation of the first amplification, IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included, for example, during step B of the process described herein, according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step B.

In some embodiments, step B of initiating the first amplification (e.g., according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

1. Feeder cells and antigen-presenting cells

In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added during the initiation of the first amplification. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5-7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 6–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 6-7 of the initiation first amplification period. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 or 8 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 8 of the initiation of the first amplification.

In some embodiments, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8B) and those amplifications referred to as pre-REP or initiation REP) requires feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification and during the initiation of the first amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, 2.5 × ^10⁸ feeder cells are used during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per container during the initiation of the first amplification. In some embodiments, 2.5 × ^{10⁸ feeder cells are used per GREX-10} during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per GREX-100 during the initiation of the first amplification.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 has not increased compared to the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells. In other embodiments, the number of feeder cells required to initiate the first expansion is one-quarter, one-third, five-twelfths, or one-half of the number of feeder cells used for rapid second expansion.

In some embodiments, the culture medium for initiating the first expansion contains IL-2. In some embodiments, the culture medium for initiating the first expansion contains 6000 IU/mL IL-2. In some embodiments, the culture medium for initiating the first expansion contains antigen-presenting feeder cells. In some embodiments, the culture medium for initiating the first expansion contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium for initiating the first expansion contains OKT-3. In some embodiments, the culture medium contains 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells. In some embodiments, the culture medium comprises 500 mL of culture medium and ¹⁵ μg OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium comprises 500 mL of culture medium, 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the initiation of the first expansion procedure described herein requires an excess of feeder cells beyond the TIL during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs to initiate the first expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is also possible to initiate the first expansion of TILs using cytokines in combination with two or more of IL-2, IL-15, and IL-21 as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly beneficial for the generation of lymphocytes, and especially T cells as described therein. See, for example, Table 2.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives may be used in the culture medium during step B, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Initiating the transition from the first amplification to the rapid second amplification

In some cases, a population of primary TILs obtained from an initial first amplification (which may include amplification sometimes referred to as pre-REP), including, for example, a population of TILs obtained from step B as indicated in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), may undergo a rapid second amplification (which may include amplification sometimes referred to as a rapid amplification protocol (REP)) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs are intended for therapeutic use, an amplified TIL population from an initial first amplification or an amplified TIL population from a rapid second amplification may be genetically modified for appropriate treatment before the amplification step or after the initial first amplification and before the rapid second amplification.

In some implementations, the TIL obtained from step B as indicated in the initiation of the first amplification (e.g., Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is stored until the phenotype is determined for selection. In some embodiments, the TIL obtained from the initiation of the first amplification (e.g., step B as indicated in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is not stored and is directly subjected to rapid second amplification. In some embodiments, the TIL obtained from the initiation of the first amplification is not cryopreserved after the initiation of the first amplification and before rapid second amplification. In some embodiments, the transition from the initiation of the first amplification to the second amplification occurs approximately 2, 3, 4, 5, 6, 7, or 8 days after tumor fragmentation occurs and/or after the initiation of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 8 days after fragmentation and/or after the start of the first initiation amplification step.

In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1, 2, 3, 4, 5, 6, 7, or 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 6 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 6 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 8 days after fragmentation and/or the start of the first initiation amplification step.

In some embodiments, TILs are not stored after the initial first amplification and before the rapid second amplification, and the TILs undergo rapid second amplification directly (e.g., in some embodiments, they are not stored during the transition from step B to step D as shown in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from which the first amplification was initiated (the second TIL population) undergo rapid second amplification directly without a transition period.

In some embodiments, the transition from initiating the first amplification to the rapid second amplification (e.g., step C according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a GREX-10 or GREX-100. In some embodiments, the closed-system bioreactor is a single bioreactor. In some embodiments, the transition from initiating the first amplification to the rapid second amplification involves a longitudinal scaling up of the container size. In some embodiments, the initiation of the first amplification is carried out in a smaller container compared to the rapid second amplification. In some implementations, the initial amplification is performed in a GREX-100 and the rapid second amplification is performed in a GREX-500.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

In view of this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from Miltenyi Biotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or Figures 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing process also includes a quiescent step. According to some implementations, the quiescent step involves incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Rapid Second Amplification

In some implementations, the TIL cell population is further expanded in number after the collection and initiation of the first expansion (steps A and B) and the transition referred to as step C, as indicated in Figures 8 (in particular, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). This further amplification, referred to herein as rapid second amplification or rapid amplification, may include an amplification process commonly referred to in the art as a rapid amplification procedure (rapid amplification protocol or REP); and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Rapid second amplification is typically performed in a ventilated container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies. In some embodiments, the TIL is transferred to a larger volume container 1, 2, 3, or 4 days after the initiation of rapid second amplification (i.e., on days 8, 9, 10, or 11 of the overall Gen 3 process).

In some embodiments, the rapid second amplification of TIL (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 9 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 1 day after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 2 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 3 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 4 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 5 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 6 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 8 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 9 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 10 days after the initiation of rapid second amplification.

In some embodiments, the rapid second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the process indicated in step D of FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells, wherein feeder cells are added to a final concentration that is two, 2.4, 2.5, 3, 3.5, or 4 times the feeder cell concentration present in the initiating first amplification. For example, TILs can be rapidly expanded by stimulation with nonspecific T cell receptors in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as approximately 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Laritan, NJ or Miltenyi Biotech, Auburn, California), or UHCT-1 (available from BioLegend, San Diego, California). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation with the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL to 60 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3. In some embodiments, the cell culture medium comprises about 60 ng/mL of OKT-3. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium in the rapid second amplification contains 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 500 mL of culture medium and 6000 IU/mL IL-2, 30 μg of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium contains 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells. In some implementations, the culture medium in the rapid second amplification comprises 500 mL of culture medium per container and 6000 IU/mL IL-2, 30 μg OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, during the second amplification period, including, for example, during step D of the process described herein according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step D.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen-presenting cells in rapid expansion and/or secondary expansion is approximately 1:10, approximately 1:15, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:75, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs in rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some implementations, the ratio of TIL to PBMC in rapid amplification and/or secondary amplification is between 1:100 and 1:200.

In some implementations, the REP and/or rapid second amplification are performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 ng/mL OKT3 anti-CD3 antibody, and 6000 IU/mL IL-2 in 150 mL of culture medium, wherein the feeder cell concentration is at least 1.1-fold (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.8X, 2X, 2.1X, 2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, or 4.0X. The culture medium is replaced (usually by removing 2/3 of the used medium and replacing it with an equal volume of fresh medium) until the cells are transferred to a replacement growth chamber. The replacement growth chamber consists of G-REX culture flasks and aeration containers, as discussed in more detail below.

In some embodiments, the rapid second amplification (which may include a process referred to as the REP process) takes 7 to 9 days, as discussed in the examples and figures. In some embodiments, the second amplification takes 7 days. In some embodiments, the second amplification takes 8 days. In some embodiments, the second amplification takes 9 days.

In some embodiments, the second amplification (which may include an amplification referred to as REP, and the amplification mentioned in step D of Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA) with a 100 cm aerated silica bottom, 5 × ^10⁶ or 10 × ^10⁶ TILs can be taken together with PBMCs in 400 mL of supplemented 5% human AB serum at 3000 IU/mL Cells were cultured in a 50/50 medium containing IL-2 and 30 ng/mL anti-CD3 (OKT3). G-REX-100 culture flasks were incubated at 37°C in 5% _CO2 . On day 5, 250 mL of the supernatant was removed and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL precipitate was resuspended in 150 mL of fresh medium containing 5% human AB serum and 6000 IU/mL IL-2 and added back to the original GREX-100 culture flask. As TILs were continuously amplified in GREX-100 culture flasks, they could be transferred to larger culture flasks, such as GREX-500, on day 10 or 11. Cells could be collected on day 14. Cells could be collected on day 15. Cells could be collected on day 16. In some embodiments, the medium was replaced until the cells were transferred to a replacement growth chamber. In some embodiments, two-thirds of the culture medium is replaced by aspirating the used medium and replacing it with an equal volume of fresh medium. In some embodiments, alternative growth chambers include GREX culture flasks and aerated containers, as discussed more fully below. In some embodiments, the process employs different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different numbers of replicates.

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some implementations, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

In some embodiments, the specific culture media described in International Patent Application Publication No. WO 1998/030679 and U.S. Patent Application Publication No. US2002/0076747A1 (which are incorporated herein by reference) are applicable to the present invention. In the disclosures, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , ^{Cr3 +} , Ge4 ⁺ , Se4+, Br, T, ^Mn2+ , P, ^Si4+ , V5 ⁺ , Mo6 ⁺ , ^{Ni2 +, Rb+} , Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) is suitable for use in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, a rapid second amplification (including an amplification termed REP) is performed, and it further includes a step of selecting TILs with superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability can be determined using standard assays known in the art after a rapid second amplification (including amplification known as REP amplification). For example, a trypan blue exclusion assay can be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample can be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve vertical scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a first mini-culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the first mini-culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the TILs transferred from the first mini-culture is cultured in the second mini-culture for a period of approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 5 days; and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid second amplification is performed over a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid second amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid second amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid second expansion before splitting contains the same components as the cell culture medium used for rapid second expansion after splitting. In some embodiments, the cell culture medium used for rapid second expansion before splitting contains different components than the cell culture medium used for rapid second expansion after splitting.

In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

1. Feeder cells and antigen-presenting cells

In some embodiments, the rapid second amplification procedure described herein (e.g., including amplifications such as those described in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In other embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:10, approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 7.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, rapid second expansion requires twice the number of feeder cells of rapid second expansion. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 5 × ^10⁸ feeder cells. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 7.5 × ^10⁸ feeder cells. In other embodiments, the rapid second expansion requires twice (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X the number of feeder cells required to initiate the first expansion.

In some embodiments, the rapid second amplification procedure described herein requires an excess of feeder cells during the rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs. In some embodiments, PBMCs are added to the rapid second amplification at twice the concentration of PBMCs added to initiate the first amplification.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs during rapid second expansion.

2. Cytokines and other additives

The rapid second amplification method described herein typically uses a culture medium containing high doses of cytokines (particularly IL-2), as is known in the art.

Alternatively, it is possible to rapidly expand TILs using combinations of cytokines, as described in U.S. Patent Application Publication No. US2017/0107490 A1, which discloses the use of combinations of two or more IL-2, IL-15, and IL-21, the contents of which are incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having specific uses in many embodiments. The use of combinations of cytokines is particularly beneficial for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Furthermore, additives, such as peroxisome proliferator-activated receptor γ coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, may be used in the culture medium during step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

Step E: Collect TIL

Cells can be collected after the rapid second amplification step. In some embodiments, TILs are collected after one, two, three, four or more amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps (one initiating a first amplification and one rapid second amplification) provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

TILs can be collected in any suitable and aseptic manner, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the process of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some embodiments, step E according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) is performed according to the process described herein. In some embodiments, the closed system is introduced via a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described herein is employed.

In some embodiments, TILs are collected according to the methods described herein. In some embodiments, TILs between days 14 and 16 are collected using the methods described herein. In some embodiments, TILs are collected on day 14 using the methods described herein. In some embodiments, TILs are collected on day 15 using the methods described herein. In some embodiments, TILs are collected on day 16 using the methods described herein.

Step F: Final formulation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and as outlined above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, the TIL amplified using the methods of this disclosure is administered to the patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. The TIL amplified as disclosed herein can be administered via any suitable route known in the art. In some embodiments, the TIL is administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VII. Other Gen 2, Gen 3 and other TIL manufacturing process implementation plans

A. PBMC feeder cell ratio

In some embodiments, the culture medium used for the amplification methods described herein (see, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) includes an anti-CD3 antibody, such as OKT-3. The combination of anti-CD3 antibody and IL-2 induces T cell activation and cell division in TIL populations. This effect is seen in full-length antibodies as well as Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

In some implementations, the number of PBMC feeder cell layers is calculated as follows:

AT cell volume (diameter 10 μm): V = (4/3) ^πr³ = 523.6 ^μm³

B. The volume of G-REX-100(M) with a height of 40 μm (4 cells): V = (4/3) ^πr³ = 4 × ^{10¹² μm³}

C. Number of cells required to fill column B: 4 × ^10¹² μm³ ^{/ 523.6 μm³ =} 7.6 × ^{10⁸ μm³} × 0.64 = 4.86 × ^10⁸

D. The optimal number of cells to activate in 4D space: 4.86 × 10⁸ ^{/ 24} = 20.25 ^{× 10⁶}

E. Extrapolation to feeder cell and TIL numbers for G-REX-500: TIL: 100 × ^10⁶ and feeder cells: 2.5 × ^10⁹

In this calculation, the approximate number of mononuclear cells required to provide TIL activation using icosahedral geometry in a cylinder with a base of 100 ^cm² is used. The experimental result for T cell threshold activation derived from the calculation is approximately 5 × ^10⁸ , which is closely correlated with NCI experimental data, as described in Jin et al., J. Immunother. 2012, 35, 283-292. In (C), the multiplier (0.64) is the random filling density of the equivalent spheres, calculated by Jaeger and Nagel, Science, 1992, 255, 1523-3. In (D), the divisor 24 is the number of equivalent spheres or “Newton number” in 4-dimensional space that can contact similar objects, as described in Musin, Russ. Math. Surv., 2003, 58, 794-795.

In some embodiments, the number of exogenously supplied antigen-presenting feeder cells during the initiation of the first expansion is approximately half the number of exogenously supplied antigen-presenting feeder cells during the rapid second expansion. In some embodiments, the method includes initiating the first expansion in a cell culture medium containing approximately 50% fewer antigen-presenting cells compared to the cell culture medium used in the rapid second expansion.

In other embodiments, the number of exogenously supplied antigen-presenting feeder cells (APCs) during the rapid second amplification is greater than the number of exogenously supplied APCs during the initiation of the first amplification.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs during the rapid second amplification period to the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, or 2.5:1. 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately ¹ ×10⁸, 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, ^1.5 × ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8×10⁸, 1.9× ^10⁸ , 2 ^{×10⁸ ,} 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3×10⁸, 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^{10⁸ ,} 3.1× ^10⁸ , or 3.2×10⁸ ^. The number of exogenously supplied APCs during the rapid second amplification period is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × 10⁸, 3.8 × ^10⁸ , 3.9 × ^10⁸ , ^{4 × 10⁸ ,} 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^10⁸ , 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , ^4.9 × ^10⁸ , 5 × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^10⁸ , 5.3 × ^10⁸ , 5.4 ^{× 10⁸ .} 5.5× ^10⁸ , 5.6× ^10⁸ , 5.7×10⁸, 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3×10⁸, 6.4× ^10⁸ , ^6.5 × ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , ^7.2 × ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , 7.8× ^10⁸ , 7.9× ^10⁸ 8× ^10⁸ , 8.1× ^10⁸ , 8.2×10⁸, 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8×10⁸, 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , ^9.7 × ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or 1×10⁹ APCs .}

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ^1.5 × 10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ² × 10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is exactly or approximately 5 × ^10⁸ APCs.

In some embodiments, the number of APCs (including, for example, PBMCs) added on day 0 of the first expansion is approximately half the number of PBMCs added on day 7 of the first expansion (e.g., day 7 of the method). In some embodiments, the method includes adding antigen-presenting cells to a first TIL population on day 0 of the first expansion and adding antigen-presenting cells to a second TIL population on day 7, wherein the number of antigen-presenting cells added on day 0 is approximately 50% of the number of antigen-presenting cells added on day 7 of the first expansion (e.g., day 7 of the method).

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is greater than the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification.

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 2 × ^10⁶ APCs/ ^cm² to just or about 3 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density of just or about 2 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} Inoculate culture flasks with APCs/cm² at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4×10⁶ ^, or 4.5× ^{10⁶ .}

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 4.0 × ^10⁶ APCs/ ^cm² to just or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from the range of exactly or approximately 4.0 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenously supplied APCs in the rapid second amplification are at exactly or approximately 2.5 × ^10⁶ APCs/ ^cm² , 2.6 × 10⁶ APCs/ ^cm² , 2.7 × ^10⁶ APCs/ ^cm² , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , 3.2 × ^10⁶ , 3.3 × ^10⁶ , 3.4 × ^10⁶ , 3.5 × ^10⁶ , 3.6 × ^10⁶ , 3.7 × ^10⁶ , 3.8 × ^10⁶ , 3.9 × ^10⁶ , 4 × ^10⁶ , 4.1 × ^10⁶ , 4.2 × ^10⁶ , 4.3 × ^10⁶ , 4.4 × ^10⁶ , or 4.5 × ^{10⁶ .} 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8×10⁶, 4.9× ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4×10⁶, 5.5× ^10⁶ , ^{5.6×10⁶ ,} 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , 6× ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , ^6.3 × ^10⁶ , 6.4×10⁶, 6.5× ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , ^6.8 × ^10⁶ , 6.9× ^10⁶ , 7× ^10⁶ Inoculate the culture flasks at densities of 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4× ^10⁶ , or 7.5× ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} The APCs were seeded in culture flasks at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , or 4.5× ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for rapid second amplification were at exactly or approximately 2.5× ^10⁶ APCs/ ^cm² , 2.6× ^10⁶ APCs/ ^cm² , 2.7× ^10⁶ APCs/ ^cm² , 2.8× ^10⁶ , 2.9× ^10⁶ , 3× ^10⁶ , and 3.1×10⁶ ^APCs/cm². 3.2× ^10⁶ , 3.3× ^10⁶ , 3.4×10⁶, 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4×10⁶, 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , 4.5× ^10⁶ , 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8× ^10⁶ , ^4.9 × ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4× ^10⁶ , 5.5× ^10⁶ , ^5.6 × ^10⁶ APCs/cm² were inoculated into culture flasks at densities of 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , ⁶ × ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , 6.3×10⁶, 6.4× ^10⁶ , ^6.5 × ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , 6.8× ^10⁶ , 6.9×10⁶, ⁷ × ^10⁶ , 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4×10⁶, or 7.5× ^{10⁶ .}

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density selected ^from exactly or about 2.5 × ^10⁶ APCs/cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in culture flasks at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in culture flasks at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 2 × ^10⁶ APCs/ ^cm² to exactly or about 3 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied at the rapid second amplification are seeded in the culture flask at a density selected from exactly or about 4 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in the culture flask at a density of exactly or approximately 2 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density of exactly or approximately 4 × ^10⁶ APCs/ ^cm² .

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other implementations, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, etc. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1× ^10⁸ , 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4× ^10⁸ , 1.5× ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9× ^10⁸ , 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , or 3.1×10⁸ ^. The exogenous supply of APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × ^10⁸ , 3.8 × ^10⁸ , 3.9 × ^10⁸ , 4 × ^10⁸ , 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^{10⁸ ,} 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , 4.9 × ^10⁸ , ⁵ × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^{10⁸ . 8} , 5.3× ^10⁸ , 5.4× ^10⁸ , 5.5×10⁸, 5.6× ^10⁸ , 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , 7.2× ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^{10⁸ 8} , 7.8× ^10⁸ , 7.9×10⁸, ⁸ ×10⁸, 8.1× ^10⁸ , 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , 9.7× ^10⁸ , ^9.8 × ^10⁸ , 9.9× ^10⁸ , or 1× ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 1 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 1 × ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 7.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 2 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 4.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 5.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or about 5 × ^10⁸ APCs (including, for example, PBMCs).

In some embodiments, the number of APC (including, for example, PBMC) layers added on day 0 of the first amplification is approximately half the number of APC (including, for example, PBMC) layers added on day 7 of the rapid second amplification. In some embodiments, the method includes adding antigen-presenting cell layers to a first TIL population on day 0 of the first amplification and adding antigen-presenting cell layers to a second TIL population on day 7, wherein the number of antigen-presenting cell layers added on day 0 is approximately 50% of the number of antigen-presenting cell layers added on day 7.

In other implementations, the number of exogenously supplied APC (including, for example, PBMC) layers on day 7 of the rapid second amplification is greater than the number of exogenously supplied APC (including, for example, PBMC) layers on day 0 of the initiation of the first amplification.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about three cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1.5 to just or about 2.5 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about two cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers, and day 7 of the rapid second amplification occurs with an average thickness of just or approximately 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3. It occurs in the presence of layered APCs (including, for example, PBMCs) of 8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1 cell layer to just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 to just or about 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1, 2, or 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9, or 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is exactly or approximately 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from... Exactly or approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1 :4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.0 × ^10⁶ APCs/ ^cm² to about 4.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 2.5 × ^10⁶ APCs/ ^cm² to about 7.5 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.5 × ^10⁶ APCs/ ^cm² to about 3.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 3.5 × ^10⁶ APCs/ ^cm² to about 6.0 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 2.0 × ^10⁶ APCs/ ^cm² to about 3.0 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 4.0 × ^10⁶ APCs/ ^cm² to about 5.5 × ^10⁶ APCs/ ^cm² .

A. Optional cell culture medium components

1. Anti-CD3 antibody

In some embodiments, the culture medium used for the amplification methods described herein (including an amplification method known as REP, see, for example, Figures 1 and 8 (especially, for example, Figure 8B)) includes an anti-CD3 antibody. The combination of the anti-CD3 antibody and IL-2 induces T cell activation and cell division in a TIL population. This effect can be seen with both full-length antibodies and Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California). See Table 1.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California).

2,4-1BB (CD137) agonist

In some embodiments, the cell culture medium initiating the first amplification and/or rapid second amplification contains a TNFRSF agonist. In some embodiments, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule may contain any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The 4-1BB agonist or 4-1BB binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies); monoclonal antibodies (including full-length monoclonal antibodies); polyclonal antibodies; multispecific antibodies (e.g., bispecific antibodies); human, humanized, or chimeric antibodies; and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to 4-1BB, such as scFv molecules. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the 4-1BB agonist used in the methods and compositions disclosed in this invention includes anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectin, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. Agonistant anti-4-1BB antibodies are known to induce strong immune responses. (Lee et al., PLOSOne 2013, 8, e69677). In some embodiments, the 4-1BB agonist is an agonistant anti-4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In some embodiments, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utumumab, or urilumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

In some embodiments, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In some embodiments, polymeric 4-1BB agonists, such as trimeric or hexameric 4-1BB agonists (having three or six ligand-binding domains), can induce the formation of desirable receptor (4-1BBL) clusters and internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins containing three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic 4-1BB antibodies and fusion proteins induce strong immune responses. In some embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO:40) with high affinity and agonistic activity. In some embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO:40). In some embodiments, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO:41). The amino acid sequences of the 4-1BB antigens bound by the 4-1BB agonist or binding molecule are summarized in Table 5.

Table 5.4-1 shows the amino acid sequence of the BB antigen.

In some embodiments, the described compositions, processes, and methods include a 4-1BB agonist that binds to human or rodent 4-1BB at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at _{a KD} of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include 4-1BB agonists that _bind to human or rodent 4-1BB at a k-association rate of about 7.5 × ^{10⁵ 1} /M·s or faster, at a k _-association rate of about 7.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ 1/M·s or faster, at a k- _association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include binding to human or rodent 4-1BB at a k _{-dissociation} rate of about 2 × ^10⁻⁵ 1/s or _slower , at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about _2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 × ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/s or _slower , at about 2.6 × ^10⁻⁵ 1/s or _slower , or at about 2.7 × ^10⁻⁵ 1/s or slower. 4-1BB agonists that bind to human or rodent 4-1BB at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include a _4-1BB agonist that binds to human or rodent 4-1BB at an _IC50 of about ₁₀ nM or less, at about 9 nM or less, at about ₈ nM or less, at about 7 nM or less, at about ₆ nM or less, at about ₅ nM or less, at about ₄ nM or less, at about 3 nM or less, at about ₂ nM or less, or at about 1 nM or less _{. 50} combines with 4-1BB in humans or rodents.

In some embodiments, the 4-1BB agonist is utumumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant, or biosimilar thereof. Utumumab is available from Pfizer. Utumumab is an immunoglobulin G2-λ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of utumumab is illustrated in Table 6. Utumumab contains glycosylation sites at Asn59 and Asn292; intrachain disulfide bridges in the heavy chain at positions 22-96 ( _VH - _VL ), 143-199 ( _CH1 - _CL ), 256-316 ( _CH2 ), and 362-420 ( _CH3 ); and intrachain disulfide bridges at positions 22'-87' ( _VH - _VL ) and 136'-195' ( _CH1 -CL _). Intrachain disulfide bridges of the light chain; interchain heavy chain-heavy chain disulfide bridges located at IgG2A isoform positions 218-218, 219-219, 222-222 and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222 and 225-225 and at IgG2B isoform positions 219-130(2), 222-222 and 225-225; and interchain heavy chain-light chain disulfide bridges located at IgG2A isoform positions 130-213'(2), IgG2A/B isoform positions 218-213' and 130-213' and at IgG2B isoform positions 218-213'(2). The preparation and characterization of utumumab, as well as its variants and fragments, are described in U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678 and International Patent Application Publication No. WO 2012/032433 A1, the disclosures of which are incorporated herein by reference. The preclinical characteristics of utumumab are described in Fisher et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utumumab in various hematologic and solid tumor indications include US National Institutes of Health (NIH) clinicaltrials.gov identification numbers NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:42 and the light chain provided in SEQ ID NO:43. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utumumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:44, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:45, along with its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of utumumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity, such as 97%, 98%, 99%, or 100%, with an amino acid sequence that is 97%, 98%, 99%, or 100% sequence identity with a reference drug or reference biological product, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, which is utumumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, which is utumumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab.

Table 6. Amino acid sequences of 4-1BB agonist antibodies associated with utumumab.

In some embodiments, the 4-1BB agonist is the monoclonal antibody urogenumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biosimilar thereof. Ureogenumab is available from Bristol-Myers Squibb and Creative Biolabs. Ureogenumab is an immunoglobulin G4-κ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of urogenumab is illustrated in Table 7. Urelimab contains N-glycosylation sites at positions 298 (and 298”); intrachain disulfide bridges in the heavy chain at positions 22–95 ( _VH – _VL ), 148–204 ( _CH1 – _CL ), 262–322 ( _CH2 ), and 368–426 ( _CH3 ) (and at positions 22”–95”, 148”–204”, 262”–322”, and 368”–426”); and intrachain disulfide bridges in the heavy chain at positions 23”–88” ( _VH – _VL ) and 136”–196” ( _CH1 –CL _). (and intra-chain disulfide bridges at positions 23”'-88”' and 136”'-196”'); inter-chain heavy chain-heavy chain disulfide bridges at positions 227-227” and 230-230”; and inter-chain heavy chain-light chain disulfide bridges at positions 135-216”' and 135”-216”'. The preparation and characterization of urogenumab, as well as its variants and fragments, are described in U.S. Patent Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical characteristics of urogenumab are described in Segal et al., Clin. Cancer. Res. 2016, available at http://dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical trials of ureglucumab in various hematologic and solid tumor indications include NCT01775631, NCT02110082, NCT02253992, and NCT01471210 from the National Institutes of Health (NIH) clinicaltrials.gov.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:52 and the light chain provided in SEQ ID NO:53. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urinumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:54, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:55, and its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of urogenumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is uroreglumab.

Table 7. Amino acid sequences of 4-1BB agonist antibodies associated with urilumab.

In some embodiments, the 4-1BB agonist is selected from the group consisting of: 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), produced by a cell line registered as ATCC No. HB-11248 and disclosed in U.S. Patent No. 6,974,863. Antibodies disclosed in the following countries include: 5F4 (BioLegend 31 1503), C65-485 (BDPharmingen 559446), antibodies disclosed in US Patent Application Publication No. 2005/0095244, antibodies disclosed in US Patent No. 7,288,638 (e.g., 20H4.9-IgG1 (BMS-663031)), antibodies disclosed in US Patent No. 6,887,673 (e.g., 4E9 or BMS-554271), and antibodies disclosed in US Patent No. 7,214,493. Antibodies disclosed in U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, U.S. Patent No. 6,905,685 (e.g., 4E9 or BMS-554271), U.S. Patent No. 6,362,325 (e.g., 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), U.S. Patent No. 6,974,863 (e.g., 53A2), and U.S. Patent No. 6,210,669 (e.g., 5E9 or 5E2). The antibodies described in U.S. Patent No. 5,928,893 (1D8, 3B8, or 3E1), U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, and International Patent Application Publications Nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, as well as fragments, derivatives, conjugates, variants, or bioanalytes thereof, wherein the disclosure of each of the foregoing patents or patent applications is incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein described in the following: International Patent Application Publications No. WO 2008/025516 A1, WO 2009/007120A1, WO 2010/003766 A1, WO 2010/010051 A1 and WO 2010/078966 A1; U.S. Patent Application Publication No. US 201 The disclosures of U.S. Patent Nos. 1/0027218A1, 2015/0126709 A1, 2011/0111494A1, 2015/0110734 A1, and 2015/0126710 A1; and U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, are incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic fusion protein as depicted in structure IA (C-terminal Fc antibody fragment fusion protein) or structure IB (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof (see Figure 18). In structures IA and IB, the cylinder refers to an individual polypeptide-binding domain. Structures IA and IB contain three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL (4-1BB ligand, CD137 ligand (CD137L), or tumor necrosis factor superfamily member 9 (TNFSF9)) or antibodies binding to 4-1BB. These TNFRSF binding domains fold to form a trivalent protein, which is then linked to a second trivalent protein via an IgG1-Fc (including _CH3 and _CH2 domains). The IgG1-Fc then serves to link the two trivalent proteins together via disulfide bonds (elongated ellipses), thereby stabilizing the structure and providing an agonist capable of linking the intracellular signaling domains of six receptors to signaling proteins to form a signaling complex. A TNFRSF binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys. Any scFv domain design can be used, such as those described in: de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad et al., Clin. & Dev. Immunol. 2012, 980250; Monnier et al., Antibodies, 2013, 2, 193-208; or other references incorporated herein by reference. The structure of this form of fusion protein is described in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of structures I-A provided in Figure 18 are shown in Table 8. The Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO: 62), a complete hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). Preferred adapters for linking C-terminal Fc antibodies are selected from the embodiments provided in SEQ ID NO: 63 to SEQ ID NO: 72, including adapters suitable for fusing other polypeptides.

Table 8. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with C-terminal Fc-antibody fragment fusion protein design (structure I-A).

The amino acid sequences of the other polypeptide domains of structures I-B provided in Figure 18 are shown in Table 9. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SED ID NO:74 to SEQ ID NO:76.

Table 9. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with N-terminal Fc-antibody fragment fusion protein design (structures I-B).

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B includes one or more 4-1BB binding domains selected from the group consisting of: variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains selected from the variable heavy and variable light chains described in Table 10, any combination of the aforementioned variable heavy and variable light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:77. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a soluble 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:78.

In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:43 and SEQ ID NO:44, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the _VH and _VL sequences provided in Table 10, wherein the _VH and _VL domains are connected by linkers.

Table 10. Applicable to the 4-1BB binding domain in fusion proteins or other polypeptide domains applicable to scFv 4-1BB agonist antibodies.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stem region (which promotes trimerization and provides a distance from the cell membrane, but is not part of the 4-1BB binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some embodiments, the 4-1BB agonist is a BPS Bioscience 4-1BB agonist antibody, catalog number 79097-2, available from BPS Bioscience in San Diego, California, USA. In other embodiments, the 4-1BB agonist is a Creative Biolabs 4-1BB agonist antibody, catalog number MOM-18179, available from Creative Biolabs in Shirley, NY, USA.

3. OX40 (CD134) agonist

In some embodiments, the TNFRSF agonist is an OX40 (CD134) agonist. The OX40 agonist can be any OX40-binding molecule known in the art. The OX40-binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX40. The OX40 agonist or OX40-binding molecule may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The OX40 agonist or OX40-binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized or chimeric antibodies, and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to OX40, such as scFv molecules. In some embodiments, the OX40 agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the OX40 agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the OX40 agonist used in the methods and compositions of this disclosure includes anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 adenettin, anti-OX40 domain antibodies, single-chain anti-OX40 fragments, heavy-chain anti-OX40 fragments, light-chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. In some embodiments, the OX40 agonist is an agonistic anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

In some embodiments, the OX40 agonist or OX40 binding molecule may also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun et al., J. Immunother. 2009, 182, 1481-89. In some embodiments, poly-OX40 agonists, such as trimeric or hexameric OX40 agonists (having three or six ligand-binding domains), can induce clustering of the preferred receptor (OX40L) and the formation of internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic OX40 antibodies and fusion proteins can induce strong immune responses. (Curti et al., Cancer Res. 2013, 73, 7189-98). In some embodiments, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficiently reducing toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC), such as NK cell toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the OX40 agonist is characterized by binding to human OX40 (SEQ ID NO: 85) with high affinity and agonistic activity. In some embodiments, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 85). In some embodiments, the OX40 agonist is a binding molecule that binds to mouse OX40 (SEQ ID NO: 86). Table 11 summarizes the amino acid sequences of the OX40 antigen bound to the OX40 agonist or binding molecule.

Table 11. Amino acid sequence of OX40 antigen.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind human or rodent OX40 at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at a _KD of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that _bind to human or rodent OX40 at a k-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at a k _{-dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about 2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 _× ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/ _s or slower, and at about 2.6 × ^10⁻⁵ 1/s or slower. It binds to human or rodent OX40 at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.7 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at an _IC50 of about 10 nM or less, at an IC50 of about 9 nM or less, at an _IC50 of about 8 nM or less _, at an _IC50 of about 7 nM or less, at an IC50 of about 6 nM or less, at an _IC50 of about ₅ nM or less, at an IC50 of about 4 nM or less, at an _IC50 of about 3 nM or less, at _{an IC50} of about 2 nM or less, or at an _IC50 of about 1 nM or less.

In some implementations, the OX40 agonist is tavoricumab, also known as MEDI 0562 or MEDI-0562. Tavoricumab is available from MedImmune, a subsidiary of AstraZeneca. Tavoricumab is a humanized and chimeric monoclonal antibody against immunoglobulin G1-κ [Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)]. The amino acid sequence of tavoricumab is illustrated in Table 12. Tavocitumab contains N-glycosylation sites at positions 301 and 301", with fucosylated complex biantennary CHO-type glycans; intrachain disulfide bridges in the heavy chain at positions 22-95 ( _VH - _VL ), 148-204 ( _CH1 - _CL ), 265-325 ( _CH2 ), and 371-429 ( _CH3 ) (and at positions 22"-95", 148"-204", 265"-325" and 371"-429"); and intrachain disulfide bridges in the heavy chain at positions 23"-88" ( _VH - _VL ) and 134"-194" ( _CH1 -CL ₎ . Intra-chain disulfide bridges of the light chain at positions 23”'-88”' and 134”'-194”'; inter-chain heavy chain-heavy chain disulfide bridges at positions 230-230” and 233-233”'; and inter-chain heavy chain-light chain disulfide bridges at positions 224-214”' and 224”-214”'. Current clinical trials of tavoricumab in various solid tumor indications include NCT02318394 and NCT02705482 from the National Institutes of Health clinicaltrials.gov.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:87 and the light chain provided in SEQ ID NO:88. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of tavoricumab. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:89, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:90, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90 _, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises an scFv antibody, said scFv antibody comprising _VH and _VL regions _, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of the OX40 agonist approved by a regulatory agency, referencing tavoricumab. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab.

Table 12. Amino acid sequences of OX40 agonist antibodies associated with tavoricumab.

In some embodiments, the OX40 agonist is 11D4, a fully human antibody available from Pfizer. The preparation and characterization of 11D4 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is set forth in Table 13.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:97 and the light chain provided in SEQ ID NO:98. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:99, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:100, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100 _, respectively. In some embodiments, the OX40 agonist comprises _VH and VL _regions , each at least 97% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody approved by a regulatory agency with reference to 11D4. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug product or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 11D4.

Table 13. Amino acid sequences of OX40 agonist antibodies associated with 11D4.

In some embodiments, the OX40 agonist is 18D8, a fully human antibody available from Pfizer. The preparation and characterization of 18D8 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is set forth in Table 14.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:107 and the light chain provided in SEQ ID NO:108. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively.

In some embodiments, the OX40 agonist comprises a heavy chain and a light chain CDR or variable region (VR) of 18D8. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:109, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:110, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO: ₁₁₀ , respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to 18D8. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence having at least 97%, such as 97%, 98%, 99%, or 100%, of a reference drug product or reference biological product, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 18D8.

Table 14. Amino acid sequences of OX40 agonist antibodies associated with 18D8.

In some embodiments, the OX40 agonist is Hu119-122, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu119-122 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is set forth in Table 15.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:117, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:118 and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu119-122. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122.

Table 15. Amino acid sequences of OX40 agonist antibodies associated with Hu119-122.

In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu106-222 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is set forth in Table 16.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:125, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:126, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 96% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu106-222. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222.

Table 16. Amino acid sequences of OX40 agonist antibodies associated with Hu106-222.

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B12). MEDI6469 is a murine monoclonal antibody. (Weinberg et al., J. Immunother. 2006, 29, 575-585). In some embodiments, the OX40 agonist is an antibody produced from a 9B12 hybridoma and hosted by Biovest Corporation (Malvern, MA, USA), as described in Weinberg et al., J. Immunother. 2006, 29, 575-585, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the antibody comprises the CDR sequence of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of MEDI6469.

In some embodiments, the OX40 agonist is L106 BD (Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the CDR of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the CDR of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some implementations, the OX40 agonist is a murine monoclonal antibody anti-mCD134/mOX40 (clone OX86), available from BioXcell's InVivoMAb in West Lebanon, New Hampshire.

In some embodiments, the OX40 agonist is selected from the OX40 agonists described below: International Patent Application Publications WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191 and WO 2014/148895; European Patent Application EP 0672141; US Patent Application Publication US2010/136030, ... US Nos. 2014/377284, 2015/190506, and 2015/132288 (including clones 20E5 and 12H3); and US U.S. Patents 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the OX40 agonist is an OX40 agonistic fusion protein as depicted in structures I-A (C-terminal Fc-antibody fragment fusion protein) or I-B (N-terminal Fc-antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof. The characteristics of structures I-A and I-B have been described above and in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequences of the polypeptide domains of structure I-A provided in Figure 18 are shown in Table 9. The Fc domain preferably comprises an intact constant domain (amino acids 17-230 of SEQ ID NO: 62), an intact hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). The preferred adapter for linking the C-terminal Fc antibody can be selected from the embodiments provided in SEQ ID NO:63 to SEQ ID NO:72, including adapters suitable for fusing other peptides. Similarly, the amino acid sequences of the polypeptide domains of structures I-B provided in Figure 18 are shown in Table 10. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SEQ ID NO:74 to SEQ ID NO:76.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40 binding domains selected from the group consisting of: the variable heavy and light chains of tavocisumab, the variable heavy and light chains of 11D4, the variable heavy and light chains of 18D8, the variable heavy and light chains of Hu119-122, the variable heavy and light chains of Hu106-222, the variable heavy and light chains selected from the variable heavy and light chains described in Table 17, any combination of the aforementioned variable heavy and light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 133. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the soluble OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 134. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 135.

In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:127 and SEQ ID NO:128, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO: 125 and SEQ ID NO: 126, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and VL regions, respectively, that are at least 95% identical to the _VH and _VL sequences provided in Table 17, wherein the _VH and _{VL regions} are connected by linkers.

Table 17. OX40 binding domains suitable for use as fusion proteins (e.g., structures I-A and I-B) or other polypeptide domains suitable for use as scFv OX40 agonist antibodies.

In some embodiments, the OX40 agonist is an OX40-agonistic single-chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain further comprising an additional domain at an N-terminus and/or a C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble OX40-binding domain; (ii) a first peptide linker; (iii) a second soluble OX40-binding domain; (iv) a second peptide linker; and (v) a third soluble OX40-binding domain further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domains are Fab or Fc fragment domains, wherein each of the soluble OX40-binding domains lacks a stem region (which facilitates trimerization and provides a distance from the cell membrane, but is not part of the OX40-binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain; (ii) a first peptide linker; (iii) a second soluble TNF superfamily cytokine domain; (iv) a second peptide linker; and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily cytokine domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonist fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In some embodiments, the OX40 agonist is an OX40-agonistic scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some implementations, the OX40 agonist is Creative Biolabs' OX40 agonist monoclonal antibody MOM-18455, which is available from Creative Biolabs in Sherry, New York, USA.

In some implementations, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, which is available from BioLegend, Inc., San Diego, California, USA.

B. Optional cell viability analysis

Optionally, after initiating the first expansion (sometimes referred to as initial bulk expansion), cell viability can be determined using standard assays known in the art. Therefore, in some embodiments, the method includes performing cell viability determination after initiating the first expansion. For example, a trypan blue exclusion assay can be performed on the bulk TIL sample, which selectively labels dead cells and allows for viability assessment. Other assays used to test viability may include (but are not limited to) an alamar blue assay and an MTT assay.

1. Cell counting, viability, and flow cytometry

In some embodiments, cell count and/or viability are measured. Expression of biomarkers (e.g., but not limited to CD3, CD4, CD8, and CD56, and any other biomarkers disclosed or described herein) can be measured by flow cytometry using a FACSCanto ^™ flow cytometer (BD Biosciences), with antibodies, such as (but not limited to) those available from BD Biosciences (BD Biosciences, San Jose, CA). Cells can be manually counted using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art (including, but not limited to, trypan blue staining). Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0282694, which is incorporated herein by reference in its entirety. Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0280436 or International Patent Application Publication No. WO/2018/081473, both of which are incorporated herein by reference in their entirety for all purposes.

In some cases, the primary TIL population can be immediately cryopreserved using the methods discussed below. Alternatively, the primary TIL population can undergo re-expansion (REP) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs will be used in therapy, the primary or REP TIL population can be genetically modified for appropriate treatment.

2. Cell Culture

In some embodiments, the method for amplifying TILs (including those discussed above and illustrated in Figures 1 and 8, particularly those exemplified in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may include the use of about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the culture medium is serum-free. In some embodiments, the culture medium used to initiate the first amplification is serum-free. In some embodiments, the culture medium used to initiate the second amplification is serum-free. In some embodiments, the culture medium used to initiate both the first and second amplifications (also referred to as rapid second amplification) is serum-free. In some embodiments, TIL number amplification uses no more than one type of cell culture medium. Any suitable cell culture medium can be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, CA). In this regard, the method of the present invention advantageously reduces the amount and number of culture media types required for TIL number amplification. In some embodiments, TIL number amplification may include feeding cells at a frequency not exceeding once every three or four days. Amplifying cell numbers in an aerated container simplifies the procedure required for cell number amplification by reducing the feeding frequency needed.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME)-free.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for a period of about 1 to 8 days, for example, about 7 days as the initiation of the first amplification, or about 8 days as the initiation of the first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for a period of about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for about 1 to 7 days (e.g., about 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TILs to a second ventilated container and amplifying the number of TILs in the second ventilated container for about 7 to 14 days or about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days, about 10 days, or about 11 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for approximately 1 to 7 days (e.g., approximately 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium comprising IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for approximately 7 to 11 days (e.g., approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, or approximately 11 days), the second ventilated container containing a cell culture medium comprising IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, TILs are amplified in a breathable container. Breathable containers have been used to amplify TILs, using PBMCs, and employing methods, compositions, and apparatus known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In some embodiments, TILs are amplified in a breathable bag. In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the Xuri Cell Expansion System W25 (GE Healthcare)). In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the WAVE bioreactor system, also known as the Xuri Cell Expansion System W5 (GE Healthcare)). In some embodiments, the cell expansion system includes a breathable cell bag with a volume selected from the group consisting of: about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

In some embodiments, TILs can be expanded in G-REX culture flasks (available from Wilson Wolf Manufacturing). Such embodiments expand the cell population from approximately 5 × ^10⁵ cells/cm² to 10 × ^10⁶ to 30 × ^10⁶ cells/cm². In some embodiments, this is not performed feedering. In some embodiments, this is not performed feedering, as long as the culture medium in the G-REX culture flask is at a height of approximately 10 cm. In some embodiments, this is not performed feedering, but one or more cytokines are added. In some embodiments, the cytokines are added as a bolus, without needing to be mixed with the culture medium. Such containers, apparatuses, and methods are known in the art and have been used to amplify TILs, and include those described in the following: U.S. Patent Application Publication No. US2014/0377739A1, International Publication No. WO 2014/210036A1, U.S. Patent Application Publication No. US 2013/0115617A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Patent No. US 8,809,050 B2, International Publication No. WO 2011/072088A2, U.S. Patent Application Publication No. US2016/0208216A1, U.S. Patent Application Publication No. US2012/0244133 A1, International Publication No. WO 2012/129201A1, and U.S. Patent Application Publication No. US2013/0102075. The disclosures of U.S. Patent No. A1, U.S. Patent No. 8,956,860 B2, International Publication No. WO 2013/173835A1, and U.S. Patent Application Publication No. US2015/0175966 A1 are incorporated herein by reference. Similar processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

Optional gene knockdown or gene knockout in C.TIL

In some embodiments, the amplified TILs of the present invention are further manipulated before, during, or after the amplification step, including during a closed, aseptic manufacturing process (each as provided herein), to temporarily alter protein expression. In some embodiments, the temporarily altered protein expression is due to temporary gene editing. In some embodiments, the amplified TILs of the present invention are treated with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in TILs. In some embodiments, TFs and/or other molecules capable of temporarily altering protein expression provide altered tumor antigen expression and/or altered number of tumor antigen-specific T cells in the TIL population.

In some embodiments, the method includes gene editing of a TIL population. In some embodiments, the method includes gene editing of a first TIL population, a second TIL population, and/or a third TIL population.

In some embodiments, the invention includes gene editing via nucleotide insertion, such as via ribonucleic acid (RNA) insertion, including the insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA) into a TIL population to promote or inhibit the expression of one or more proteins, as well as a combination of promoting one group of proteins and inhibiting another group of proteins.

In some embodiments, the amplified TILs of the present invention undergo a temporary alteration of protein expression. In some embodiments, the temporary alteration of protein expression occurs in the host TIL population prior to the first amplification, including, for example, the TIL population obtained from step A as indicated in, for example, Figure 8 (particularly Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs during the first amplification, including, for example, the TIL population obtained from step B as indicated in Figure 8 (e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs after the first amplification, including, for example, the TIL population transitioning between the first and second amplifications (e.g., the second TIL population described herein), the TIL population obtained from, for example, step B as indicated in Figure 8, and included in step C. In some embodiments, the temporary alteration of protein expression occurs in the main TIL population prior to the second amplification, including, for example, the TIL population obtained from, for example, step C as indicated in Figure 8, and prior to its amplification in step D. In some embodiments, the temporary alteration of protein expression occurs during a second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8 (e.g., a third TIL population). In some embodiments, the temporary alteration of protein expression occurs after the second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8.

In some embodiments, the method for temporarily altering protein expression in a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. In some embodiments, the method for temporarily altering protein expression in a TIL population includes a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include transfection steps using methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

In some embodiments, temporary alteration of protein expression induces an increase in stem memory T cells (TSCMs). TSCMs are early progenitor cells of antigen-experiencing central memory T cells. TSCMs typically exhibit the long-term survival, self-renewal, and pluripotent capabilities that define stem cells and are generally required for the production of effective TIL products. In mouse models of adoptive cell transfer, enhanced antitumor activity of TSCMs compared to other subsets of T cells has been demonstrated. In some embodiments, temporary alteration of protein expression induces a TIL population with a composition containing a high proportion of TSCMs. In some embodiments, temporary alteration of protein expression induces an increase in the percentage of TSCMs of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, a temporary alteration of protein expression causes a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCM in a TIL population. In some embodiments, a temporary alteration of protein expression causes a TIL population with at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM. In some implementations, a temporary alteration in protein expression results in a therapeutic TIL population having at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM.

In some implementations, a temporary alteration of protein expression induces antigen-experiencing T-cell rejuvenation. In some implementations, rejuvenation includes, for example, increased proliferation, increased T-cell activation, and/or increased antigen recognition.

In some implementations, temporarily altering protein expression changes the expression of a large portion of T cells to preserve the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression does not alter the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression maintains the tumor-derived TCR reservoir.

In some implementations, temporary alteration of a protein induces a change in the expression of a specific gene. In some implementations, the temporary alteration of protein expression targets genes including (but not limited to) the following: PD-1 (also known as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, chimeric co-stimulatory receptor (CCR), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, and CC. R5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some implementations, protein expression targeting genes selected from the following groups is temporarily altered: PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL. 2(MCP-1), CCL3(MIP-1α), CCL4(MIP1-β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some embodiments, protein expression is temporarily altered to target PD-1. In some embodiments, protein expression is temporarily altered to target TGFBR2. In some embodiments, protein expression is temporarily altered to target CCR4/5. In some embodiments, protein expression is temporarily altered to target CBL-B. In some embodiments, protein expression is temporarily altered to target CISH. In some embodiments, protein expression is temporarily altered to target CCR (chimeric co-stimulatory receptor). In some embodiments, protein expression is temporarily altered to target IL-2. In some embodiments, protein expression is temporarily altered to target IL-12. In some embodiments, protein expression targeting IL-15 is temporarily altered. In some embodiments, protein expression targeting IL-21 is temporarily altered. In some embodiments, protein expression targeting NOTCH 1/2 ICD is temporarily altered. In some embodiments, protein expression targeting TIM3 is temporarily altered. In some embodiments, protein expression targeting LAG3 is temporarily altered. In some embodiments, protein expression targeting TIGIT is temporarily altered. In some embodiments, protein expression targeting TGFβ is temporarily altered. In some embodiments, protein expression targeting CCR1 is temporarily altered. In some embodiments, protein expression targeting CCR2 is temporarily altered. In some embodiments, protein expression targeting CCR4 is temporarily altered. In some embodiments, protein expression targeting CCR5 is temporarily altered. In some embodiments, protein expression targeting CXCR1 is temporarily altered. In some embodiments, protein expression targeting CXCR2 is temporarily altered. In some embodiments, protein expression targeting CSCR3 is temporarily altered. In some embodiments, protein expression targeting CCL2 (MCP-1) is temporarily altered. In some embodiments, protein expression targeting CCL3 (MIP-1α) is temporarily altered. In some embodiments, protein expression targeting CCL4 (MIP1-β) is temporarily altered. In some embodiments, protein expression targeting CCL5 (RANTES) is temporarily altered. In some embodiments, protein expression targeting CXCL1 is temporarily altered. In some embodiments, protein expression targeting CXCL8 is temporarily altered. In some embodiments, protein expression targeting CCL22 is temporarily altered. In some embodiments, protein expression targeting CCL17 is temporarily altered. In some embodiments, protein expression targeting VHL is temporarily altered. In some embodiments, protein expression targeting CD44 is temporarily altered. In some embodiments, protein expression targeting PIK3CD is temporarily altered. In some embodiments, protein expression targeting SOCS1 is temporarily altered. In some embodiments, protein expression targeting thymocyte selection-associated high migration group (HMG) box (TOX) is temporarily altered. In some embodiments, protein expression targeting ankyrin repeat domain 11 (ANKRD11) is temporarily altered. In some embodiments, protein expression targeting BCL6 co-inhibitor (BCOR) is temporarily altered. In some embodiments, protein expression targeting cAMP protein kinase A (PKA) is temporarily altered.

In some embodiments, a temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors. In some embodiments, the chemokine receptors overexpressed due to temporary protein expression include receptors with ligands, said ligands including (but not limited to) CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.

In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and/or TGFβ (including resulting in, for example, TGFβ pathway blockade). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CBLB (CBL-B). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CISH. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of TIGIT. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors to, for example, improve TIL transport or motility to tumor sites. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CCRs (chimeric co-stimulatory receptors). In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors selected from the group consisting of: CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.

In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins. In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins selected from the group consisting of IL-2, IL-12, IL-15, and/or IL-21.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of NOTCH 1/2 ICD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of VHL. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CD44. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of PIK3CD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of SOCS1.

In some implementations, temporarily altering protein expression causes a decrease and/or reduction in the expression of cAMP protein kinase A (PKA).

In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of two molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one molecule selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of the following: PD-1, CTLA-4, LAG-3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one of the following: CTLA-4, LAG3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CTLA-4. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIM-3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and PD-1. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and TIGIT.

In some implementations, adhesion molecules selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof are inserted into a first TIL population, a second TIL population, or a collected TIL population via gamma retrovirus or lentivirus methods (e.g., increased expression of adhesion molecules).

In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBL-B, and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, expression is reduced by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some implementations, expression is reduced by at least approximately 90%. In some implementations, expression is reduced by at least approximately 95%. In some implementations, expression is reduced by at least approximately 99%.

In some embodiments, expression is increased by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some implementations, expression increases by at least about 90%. In some implementations, expression increases by at least about 95%. In some implementations, expression increases by at least about 99%.

In some embodiments, the temporary alteration of protein expression is induced by treating the TIL with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in the TIL. In some embodiments, intracellular delivery of transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression is performed using a microfluidic platform without SQZ vectors. Such methods have demonstrated the ability to deliver proteins comprising transcription factors to a variety of primary human cell types, including T cells, as described in U.S. Patent Application Publications Nos. US2019/0093073A1, US2018/0201889A1, and US2019/0017072A1, the disclosures of which are each incorporated herein by reference. Such methods can be used in this invention to expose a TIL population to transcription factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression provide an increase in the expression of tumor antigens and/or the number of tumor antigen-specific T cells in the TIL population, thereby leading to TIL population reprogramming and increased therapeutic efficacy of the reprogrammed TIL population compared to the non-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in effector T cells and/or central memory T cell subsets relative to the initial or previous TIL population (i.e., prior to reprogramming), as described herein.

In some embodiments, the transcription factor (TF) includes (but is not limited to) TCF-1, NOTCH 1/2 ICD, and/or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1/2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered with an induced pluripotent stem cell culture (iPSC), such as a commercially available KNOCKOUT serum alternative (Gibco/ThermoFisher), to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered with an iPSC mixture to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is not administered with an iPSC mixture. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some implementations, reprogramming causes a percentage increase in TSCM of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% of TSCM.

In some embodiments, the method of temporarily altering protein expression as described above may be combined with a method of genetically modifying TIL populations, including a step of stabilizing the incorporation of genes for producing one or more proteins. In some embodiments, the method includes a step of genetically modifying TIL populations. In some embodiments, the method includes genetically modifying a first TIL population, a second TIL population, and/or a third TIL population. In some embodiments, the method of genetically modifying TIL populations includes a retroviral transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in: Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71; and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a gamma-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include transposases provided as DNA expression vectors or as expressible RNA or proteins, such that long-term expression of the transposase does not occur in transgenic cells, for example, transposases provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems, including salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, are described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In some implementations, temporary alteration of protein expression in the TIL is induced by small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, which is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where it interferes with the expression of specific genes having complementary nucleotide sequences.

In some implementations, the temporary alteration of protein expression is a reduction in expression. In some implementations, the temporary alteration of protein expression in the TIL is induced by self-delivered RNA interference (sdRNA), which is a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or _-OCH3 ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand bound to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence. sdRNA is a covalently and hydrophobically modified RNAi compound that does not require a delivery medium to enter the cell. sdRNA is typically an asymmetrically modified nucleic acid molecule with a very small double-stranded region. sdRNA molecules typically contain single-stranded and double-stranded regions, and various chemical modifications can be incorporated into these regions. Furthermore, as described herein, sdRNA molecules can be linked to hydrophobic conjugates such as conventional and higher sterol molecules. sdRNAs and related methods for their preparation have been extensively described, for example, in U.S. Patent Application Publications US2016/0304873 A1, US2019/0211337A1, US2009/0131360A1, and US2019/0048341A1, and U.S. Patents 10,633,654 and 10,913,948B2, the disclosures of which are each incorporated herein by reference. To optimize sdRNA structure, chemical properties, target location, sequence preference, etc., an algorithm has been developed and used for sdRNA efficacy prediction. Based on these analyses, a functional sdRNA sequence is generally defined as one whose expression is reduced by more than 70% at a concentration of 1 μM, with a probability exceeding 40%.

Double-stranded DNA (dsRNA) can generally be used to define any molecule containing a pair of complementary RNA strands, typically sense (passenger) and antisense (guide) strands, and may include single-stranded overhang regions. Unlike siRNA, the term dsRNA generally refers to a precursor molecule comprising the sequence of an siRNA molecule, which is released from a larger dsRNA molecule by the action of a lysin system (including Dicer).

In some implementations, the method includes temporarily altering protein expression in a population of TILs (including TILs modified to express CCR), including using self-delivered RNA interference (sdRNA), which is, for example, a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or -OCH ₃ ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand that binds to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Methods using siRNA and sdRNA have been described in the following: Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248; Byrne et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93, the disclosures of which are incorporated herein by reference. In some embodiments, siRNA delivery is accomplished using electroporation or cell membrane disruption (e.g., squeezing or SQZ). In some embodiments, delivery of siRNA or sdRNA to a TIL population does not require electroporation, SQZ, or other methods; the TIL population is actually exposed to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, the method includes delivery of siRNA or sdRNA to a TIL population, which involves exposing the TIL population to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 10 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration between 0.1 μM/10,000 TILs and 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days, wherein the exposure to siRNA or sdRNA is performed two, three, four, or five times by adding fresh siRNA or sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. Patent Application Publications Nos. US 2011/0039914A1, US 2013/0131141A1 and US 2013/0131142A1, and U.S. Patent No. 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA is inserted into the TIL population during manufacturing. In some embodiments, the sdRNA encodes RNA that interferes with the following: NOTCH 1/2 ICD, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and/or CBL-B. In some embodiments, the expression reduction is determined based on the percentage of gene silencing assessed, for example, by flow cytometry and/or qPCR. In some embodiments, the expression reduction is approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

Self-deliverable RNAi technology based on chemically modified siRNA can be used in the methods of this invention to successfully deliver sdRNA to TILs as described herein. The combination of backbone modification with asymmetric siRNA structures and hydrophobic ligands (see, for example, Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93 and U.S. Patent Application Publication No. 2016/0304873A1, the disclosure of which is incorporated herein by reference) allows sdRNA to permeate cultured mammalian cells simply by adding it to the culture medium, utilizing the nuclease stability of sdRNA without the need for additional formulations and methods. This stability allows for the support of constant levels of RNAi-mediated reduction in target gene activity simply by maintaining an effective concentration of sdRNA in the culture medium. Although not bound by theory, backbone stabilization of sdRNA provides extended reductions in gene expression that can persist for months in non-dividing cells.

In some embodiments, over 95% TIL transfection efficiency and target expression reduction occur through various specific siRNAs or sdRNAs. In some embodiments, siRNAs or sdRNAs containing several unmodified ribose residues undergo fully modified sequence substitutions to increase the potency and/or lifetime of the RNAi effect. In some embodiments, the expression reduction effect lasts for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or longer. In some embodiments, the expression reduction effect decreases after siRNA or sdRNA treatment of TILs for 10 days or longer. In some embodiments, target expression is reduced by more than 70%. In some embodiments, target expression in TILs is reduced by more than 70%. In some embodiments, expression reduction in the PD-1/PD-L1 pathway allows TILs to exhibit a more potent in vivo effect, which in some embodiments is due to avoiding the inhibitory effects of the PD-1/PD-L1 pathway. In some embodiments, TIL proliferation is increased due to reduced PD-1 expression by siRNA or sdRNA.

In some embodiments, the sdRNA sequence used in this invention exhibits a 70% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 75% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 80% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 85% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 90% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 95% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 99% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM to about 4 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 4.0 μM.

In some embodiments, the siRNA or sdRNA oligonucleotide agent comprises one or more modifications to increase the stability and/or efficacy of the therapeutic agent and to enable efficient delivery of the oligonucleotide to the cells or tissues to be treated. Such modifications may include 2'-O-methyl modifications, 2'-O-fluorine modifications, dithiophosphate modifications, 2'F-modified nucleotides, 2'-O-methyl-modified and/or 2'-deoxynucleotides. In some embodiments, the oligonucleotide is modified to include one or more hydrophobic modifications, including, for example, sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and/or phenyl. In some embodiments, the chemically modified nucleotide is a combination of thiophosphate, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and thiophosphate. In some embodiments, the sugar may be modified, and the modified sugar may include (but is not limited to) D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halogen (including 2'-fluorine), T-methoxyethoxy, 2'-allyloxy ( _-OCH₂CH = _CH₂ ), 2'-propynyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano. In some embodiments, the sugar moiety may be a hexose and incorporated into an oligonucleotide, such as Augustyns et al., Nucl. Acids. Res. 1992, 18, 4711, the disclosure of which is incorporated herein by reference.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is double-stranded along its entire length, i.e., without a dangling single-stranded sequence at either end of the molecule, i.e., a blunt end. In some embodiments, individual nucleic acid molecules may have different lengths. In other words, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is not double-stranded along its entire length. For example, when using two separate nucleic acid molecules, one of the molecules, such as the first molecule containing an antisense sequence, may be longer than the second molecule with which it hybridizes (leaving a portion of the molecule single-stranded). In some embodiments, when using a single nucleic acid molecule, a portion of the molecule at either end may remain single-stranded.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention contain mismatches and/or loops or protrusions, but are double-stranded for at least about 70% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded for at least about 80% of the oligonucleotide length. In other embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 90%-95% of the oligonucleotide length. In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 96%-98% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention contain at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

In some embodiments, siRNA or sdRNA oligonucleotides may be substantially protected against nucleases, for example, by modifying the 3' or 5' bonds, as described in U.S. Patent No. 5,849,902, the disclosure of which is incorporated herein by reference. For example, oligonucleotides may be made resistant by incorporating a “blocking group.” As used herein, the term “blocking group” refers to a substituent (e.g., other than an OH group) that can be linked to the oligonucleotide or nucleoside as a protecting or coupling group for synthesis (e.g., FITC, propyl ( _CH₂ - _CH₂ - _CH₃ ), diol (-O- _CH₂ - ^CH₂ _- O-) phosphate ( _PO₃²⁻ ), hydrogen phosphonate, or aminophosphite). A “blocking group” may also include a “terminal blocking group” or “exonuclease blocking group” that protects the 5' and 3' ends of the oligonucleotide, comprising modified nucleotide and non-nucleotide exonuclease resistant structures.

In some implementations, at least a portion of the continuous polynucleotides within the siRNA or sdRNA are linked by substituents, such as phosphate thioesters.

In some embodiments, chemical modification may result in at least 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 13% Cellular uptake of siRNA or sdRNA is enhanced at 0%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, multiple C and U residues contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U may contain hydrophobic modifications. In some embodiments, all C and U contain hydrophobic modifications.

In some embodiments, siRNA or sdRNA molecules exhibit enhanced endosome release via the incorporation of a protonable amine. In some embodiments, the protonable amine is incorporated into the sense strand (the molecular portion discarded after RISC loading). In some embodiments, the siRNA or sdRNA compounds of the present invention comprise an asymmetric compound comprising a double-stranded region (required for efficient RISC entry, 10-15 bases long) and a single-stranded region 4-12 nucleotides long; a double-stranded region having 13 nucleotides. In some embodiments, a single-stranded region of 6 nucleotides is employed. In some embodiments, the single-stranded region of the siRNA or sdRNA comprises 2-12 phosphate thioester nucleotide links (referred to as phosphate thioester modification). In some embodiments, 6-8 phosphate thioester nucleotide links are employed. In some embodiments, the siRNA or sdRNA compounds of the present invention further include unique chemical modification patterns that provide stability and compatibility with RISC entry. For example, the guide strand can also be modified by any chemical modification that demonstrates stability without interfering with RISC entry. In some implementations, the chemical modification pattern in the guide chain includes 2'F modification of most C and U nucleotides and phosphorylation at the 5' end.

In some embodiments, at least 30% of the nucleotides in the siRNA or sdRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6 3%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides are modified. In some embodiments, 100% of the nucleotides in the siRNA or sdRNA are modified.

In some embodiments, the siRNA or sdRNA molecule has a very small double-stranded region. In some embodiments, the double-stranded region of the molecule is between 8 and 15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides in length. There may be 100% complementarity between the guide strand and the passenger strand, or one or more mismatches may exist between the guide strand and the passenger strand. In some embodiments, the molecule has a blunt end or a dangling structure with one nucleotide at one end. The single-stranded region of the molecule is between 4 and 12 nucleotides in length in some embodiments. In some embodiments, the single-stranded region may be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In some embodiments, the single-stranded region may also be less than 4 nucleotides or greater than 12 nucleotides in length. In some embodiments, the single-stranded region is 6 or 7 nucleotides in length.

In some embodiments, the siRNA or sdRNA molecules exhibit increased stability. In some cases, the chemically modified siRNA or sdRNA molecules have a half-life in culture medium longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours, including any intermediate values. In some embodiments, the siRNA or sdRNA has a half-life in culture medium exceeding 12 hours.

In some embodiments, siRNA or sdRNA is optimized to increase efficacy and/or reduce toxicity. In some embodiments, the nucleotide length of the guide strand and/or passenger strand and/or the number of phosphate thioester modifications in the guide strand and/or passenger strand can affect the efficacy of the RNA molecule in some respects, while replacing 2'-fluorine (2'F) modifications with 2'-O-methyl (2'OMe) modifications can affect the toxicity of the molecule in some respects. In some embodiments, reducing the 2'F content of the molecule is expected to reduce the toxicity of the molecule. In some embodiments, the number of phosphate thioester modifications in the RNA molecule can affect the efficiency of molecule uptake into cells, for example, passive uptake of molecules into cells. In some embodiments, siRNA or sdRNA does not have 2'F modifications, but is characterized by equivalent efficacy in cellular uptake and tissue penetration.

In some embodiments, the guide chain is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate ester modifications. For example, the guide chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate ester-modified nucleotides. The guide chain may contain one or more modifications that confer increased stability without interfering with RISC entry. Phosphate-ester-modified nucleotides, such as thiophosphate-modified nucleotides, may be at the 3' end, 5' end, or throughout the entire guide chain. In some embodiments, the 3' end 10 nucleotides of the guide chain contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 thiophosphate-modified nucleotides. The guide chain may also contain 2'F and/or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at position 1 in the guide chain (the nucleotide at the 5' end of the guide chain) is modified with 2'OMe and/or phosphorylated. C and U nucleotides within the guide chain may be modified with 2'F. For example, the C and U nucleotides at positions 2-10 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'F. The C and U nucleotides within the guide chain may also be modified with 2'OMe. For example, the C and U nucleotides at positions 11-18 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'OMe. In some embodiments, the nucleotides at the 3' end of the guide chain are unmodified. In some embodiments, most of the C and U nucleotides within the guide chain are modified with 2'F, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, the 5' end of the guide chain is phosphorylated, and the C or U at positions 2-10 are modified with 2'F.

Self-deliverable RNAi technology provides a method for directly transfecting cells with an RNAi agent (whether siRNA, sdRNA, or other RNAi agents) without additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity, and ease of use are characteristics of the compositions and methods, which offer significant functional advantages over conventional siRNA-based techniques, and therefore the sdRNA method is employed in several embodiments of the method for reducing target gene expression in TILs of the present invention. The sdRNA method allows for the direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues in vitro and in vivo. The sdRNAs described in some embodiments of the invention herein are available from Advirna LLC, Worcester, MA, USA.

siRNA and sdRNA can be formed by hydrophobically modified siRNA-antisense oligonucleotide hybridization and are disclosed, for example, in Byrne et al., J. Ocular Pharmacol. Therapeut., 2013, 29, 855-864, the contents of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA oligonucleotides may be delivered to the TILs described herein using sterile electroporation. In some embodiments, the method includes sterile electroporation of TIL populations to deliver siRNA or sdRNA oligonucleotides.

In some embodiments, the oligonucleotide may be delivered to cells in combination with a transmembrane delivery system. In some embodiments, this transmembrane delivery system includes lipids, viral vectors, etc. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that does not require any delivery agent. In some embodiments, the method includes using a transmembrane delivery system to deliver siRNA or sdRNA oligonucleotides to a TIL population.

The oligonucleotides and oligonucleotide compositions are contacted (e.g., brought into contact, also referred to herein as being administered or delivered to) and taken up, including passively via the TIL. sdRNA may be added to the TIL as described herein at the following times: during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D), after step D and before collection in step E, during or after collection in step F, before or during final formulation and/or transfer to the infusion bag in step F, and before any optional cryopreservation step in step F. Furthermore, sdRNA may be added after thawing from any cryopreservation step in step F. In some embodiments, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TIL and other agents at concentrations selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM. In some implementations, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TILs and other agents in amounts selected from the group consisting of: 0.1 μM sdRNA/10,000 TILs/100 μL of medium, 0.5 μM sdRNA/10,000 TILs/100 μL of medium, 0.75 μM sdRNA/10,000 TILs/100 μL of medium. IL/100μL medium, 1μM sdRNA/10,000 TILs/100μL medium, 1.25μM sdRNA/10,000 TILs/100μL medium, 1.5μM sdRNA/10,000 TILs/100μL medium, 2μM sdRNA/10,000 TILs/100μL medium, 5μM sdRNA/10,000 TILs/100μL medium, or 10μM sdRNA/10,000 TILs/100μL medium. In some implementations, one or more sdRNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to TIL cultures twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the pre-REP or REP phase.

The oligonucleotide compositions of the present invention, including siRNA or sdRNA, can be contacted with TILs as described herein during an amplification process, for example, by dissolving high concentrations of siRNA or sdRNA in a cell culture medium and allowing sufficient time for passive uptake. In some embodiments, the method of the present invention includes contacting a TIL population with the oligonucleotide composition as described herein. In some embodiments, the method includes dissolving an oligonucleotide (e.g., siRNA or sdRNA) in a cell culture medium and contacting the cell culture medium with the TIL population. TILs may be a first, second, and/or third population as described herein.

In some embodiments, delivery of oligonucleotides into cells can be enhanced by suitable methods known in the art, including calcium phosphate, DMSO, glycerol or polydextrose, electroporation, or by transfection, for example using cationic, anionic or neutral lipid compositions or liposomes, using methods known in the art, such as those described in: U.S. Patent Nos. 4,897,355; 5,459,127; 5,631,237; 5,955,365; 5,976,567; 10,087,464; and 10,155,945; and Bergan et al., Nucl. Acids Res. 1993, 21,3567, the disclosures of which are each incorporated herein by reference.

In some embodiments, more than one siRNA or sdRNA is used to reduce the expression of a target gene. In some embodiments, one or more of PD-1, TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH that target siRNA or sdRNA are used together. In some embodiments, PD-1 siRNA or sdRNA is used with one or more of TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH to reduce the expression of more than one gene target. In some embodiments, LAG3 siRNA or sdRNA is used in combination with CISH that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, TIGIT siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CISH siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CTLA-4 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, LAG-3 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, CBL-B siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, the siRNA or sdRNA targeting one or more of PD-1, TIM-3, CBL-B, LAG3, and/or CISH described herein is available from Advirna LLC, Worcester, Massachusetts, USA, or from several other suppliers.

In some embodiments, siRNA or sdRNA targets genes selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets genes selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, the siRNA or sdRNA targets genes selected from PD-1, LAG-3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from PD-1 and one of the following: LAG3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets CISH, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets PD-1. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CBL-B.

As discussed herein, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been genetically modified by gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing of TIL populations via nucleotide insertion (RNA or DNA) to promote and inhibit the expression of one or more proteins, and combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into therapeutic populations, wherein the methods include gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, said gene editing techniques being suitable for use according to the present invention. Such methods include those described below as well as viral and transposon methods described elsewhere herein. In some embodiments, methods for genetically modifying TILs, MILs, or PBLs to express CCRs may further include modifications to inhibit gene expression via stable gene knockout or temporary gene knockdown of such genes.

In some embodiments, the method includes methods for genetically modifying TIL populations in a first, second, and/or third population as described herein. In some embodiments, the method for genetically modifying TIL populations includes a step of stabilizing a gene incorporated for producing or repressing (e.g., silencing) one or more proteins. In some embodiments, the method for genetically modifying TIL populations includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, including the step of applying a series of at least three DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time, and such that the survival rate of the TIL is maintained. In some embodiments, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for genetically modifying TIL populations include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population includes a transfection step using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference. The TIL may be a first TIL population, a second TIL population, and/or a third TIL population as described herein.

According to one embodiment, gene editing methods may include the use of programmable nucleases that mediate the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the product of the target gene.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems (e.g., Cas9) target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, the method for amplifying TILs into a therapeutic population can be performed according to any embodiment of the method described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein the method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by in vitro comparison of gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs. In some implementations, the method includes gene editing of TIL populations using CRISPR, TALE, and/or ZFN methods.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs using a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf1). According to a particular embodiment, the CRISPR method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the CRISPR method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

CRISPR stands for clustered regularly spaced short palindromic repeats. The method of gene editing using the CRISPR system is also referred to herein as the CRISPR method. There are three types of incorporating RNA and Cas proteins and CRISPR systems that can be used according to the present invention: type I, II, and III. Type II CRISPR (using Cas9 as an example) is one of the most fully characterized systems.

CRISPR technology is adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins (including Cas9) to prevent attacks from viruses and other foreign organisms by cleaving and destroying the DNA of invading foreign organisms. CRISPR is a DNA-specialized region with two unique characteristics: nucleotide repeat sequences and spacers. The nucleotide repeat sequences are distributed throughout the CRISPR region, with short foreign DNA segments (spacers) interspersed within the repeat sequences. In the type II CRISPR/Cas system, the spacers are integrated into the CRISPR genomic locus and transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNA (tracrRNA) and guide the Cas proteins to perform sequence-specific cleavage and silence pathogenic DNA. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved protospacer adjacent motif (PAM) sequence containing a dinucleotide upstream of the crRNA-binding region. Thus, the CRISPR/Cas system can retarget to cleave almost any DNA sequence by redesigning the crRNA. The crRNA and tracrRNA in the native system can be simplified to a single guide RNA (sgRNA) of approximately 100 nucleotides for genetic engineering. The CRISPR/Cas system can be directly introduced into human cells by co-delivering plasmids expressing the Cas9 endonuclease and the essential crRNA component. Different Cas protein variants can be used to reduce targeting limitations (e.g., heterologous homologs of Cas9, such as Cpf1).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via CRISPR methods include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. , CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using CRISPR methods and applicable to embodiments of the invention are described in U.S. Patents 8,697,359, 8,993,233, 8,795,965, 8,771,945, 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 8,871,445, 8,906,616, and 8,895,308, the disclosures of which are each incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are available from companies such as GenScript.

In some implementations, genetic modification of the TIL population as described herein can be performed using a CRISPR/Cpf1 system as described in U.S. Patent No. US9790490, the disclosure of which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the TALE method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the TALE method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for transcription activator-like effector protein, which includes transcription activator-like effector nucleases (TALENs). The method of gene editing using the TALE system is also referred to herein as the TALE approach. TALEs are naturally occurring proteins from the plant pathogenic bacterium *Xanthomonas* and contain a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize consecutive DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by a 14–20 base-pair spacer region, pull FokI monomers together to dimerize and generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are each incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a zinc finger or zinc finger nuclease method. According to a particular embodiment, the zinc finger method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the zinc finger method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Individual zinc fingers, exhibiting a conserved ββα conformation, contain approximately 30 amino acids. Several amino acids on the α-helix surface typically contact the 3 bp in the main groove of the DNA at varying levels of selectivity. Zinc fingers possess two protein domains. The first domain is a DNA-binding domain, which includes eukaryotic transcription factors and contains the zinc finger. The second domain is a nuclease domain, which includes the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

Individual zinc finger (ZFN) DNA-binding domains typically contain between three and six individual zinc finger repeats and can each recognize between nine and 18 base pairs. If the zinc finger domains are specific for their intended target sites, even a pair of three-finger ZFNs recognizing a total of 18 base pairs can theoretically target a single locus in the mammalian genome. One method for generating novel zinc finger arrays is to combine smaller zinc finger “modules” with known specificity. The most common module assembly process involves combining three separate zinc fingers, each recognizing a 3-base-pair DNA sequence, to produce a three-finger array recognizing a 9-base-pair target site. Alternatively, selection-based methods, such as oligopool engineering (OPEN), can be used to select new zinc finger arrays from a randomly grouped library that considers context-dependent interactions between neighboring fingers. Engineered zinc fingers are available from Sangamo Biosciences (Richmond, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the zinc finger method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, and C. ASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6 R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the zinc finger method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which can be used according to embodiments of the invention are described in U.S. Patents 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, each of which is incorporated herein by reference.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which may be used according to other embodiments of the invention are described in Beane et al., Mol. Therapy, 2015, 23, 1380-1390, the disclosure of which is incorporated herein by reference.

In some embodiments, TILs are optionally genetically engineered to include other functionalities, including (but not limited to) high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). In some embodiments, the method includes genetically engineering a population of TILs to include high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). Suitablely, the TIL population may be a first, second, and/or third population as described herein.

D. Closed systems used in TIL manufacturing

This invention provides a closed system for use during TIL (Total Intake Liquid) culture. Such a closed system allows for the prevention and/or reduction of microbial contamination, allows for the use of fewer culture flasks, and allows for lower costs. In some embodiments, the closed system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

A sterile connection device (STCD) creates a sterile weld between two compatible tubes. This procedure allows for the aseptic connection of multiple containers and tube diameters. In some embodiments, the closure system includes a Luer lock and a heat-sealing system as described in the examples. In some embodiments, the closure system is introduced under aseptic conditions via a syringe to maintain the sterility and closure properties of the system. In some embodiments, a closure system as described in the examples is used. In some embodiments, the TIL is formulated into a final product formulation container according to the method described in the examples herein.

In some embodiments, the closed system uses a single container from the time the tumor fragment is obtained until it is ready for TIL administration to the patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G container, and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. The closed system or closed TIL cell culture system is characterized by tightly sealing the system from the outside to create a closed environment free from bacterial, fungal, and/or other microbial contamination once the tumor sample and/or tumor fragment has been added.

In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

The closed system allows TILs to grow in the absence of microbial contamination and/or with significantly reduced microbial contamination.

Furthermore, the pH, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment each change with cell culture. Therefore, even if the culture medium suitable for cell culture is circulated, the closed environment still needs to be continuously maintained to provide the optimal environment for TIL proliferation. For this purpose, it is desirable to monitor the physical factors of pH, carbon dioxide partial pressure, and oxygen partial pressure within the culture medium of the closed environment using sensors. These signals are used to control a gas exchanger installed at the inlet of the culture environment, and the gas partial pressure of the closed environment is adjusted in real time according to changes in the culture medium to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system incorporating a gas exchanger equipped with monitoring devices for measuring the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment at the inlet, and optimizing the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring devices.

In some implementations, the pressure within the closed environment is controlled continuously or intermittently. That is, the pressure within the closed environment can be varied by means of, for example, a pressure maintaining device, thereby ensuring that the space is suitable for TIL growth under positive pressure or promoting exudation of fluid and thus promoting cell proliferation under negative pressure. Furthermore, by intermittently applying negative pressure, it is possible to uniformly and effectively displace the circulating fluid within the closed environment by temporarily reducing its volume.

In some implementations, the optimal culture components for TIL proliferation may be replaced or added, and factors including, for example, IL-2 and/or OKT3, and combinations thereof may be added.

E.TIL's optional cryopreservation

Optionally, the primary TIL population (e.g., a second TIL population) or an amplified TIL population (e.g., a third TIL population) can be cryopreserved. In some embodiments, therapeutic TIL populations are cryopreserved. In some embodiments, cryopreservation occurs on TILs collected after a second amplification. In some embodiments, TILs are cryopreserved in exemplary step F of Figures 1 and/or 8 (particularly, e.g., Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs are cryopreserved in infusion bags. In some embodiments, TILs are cryopreserved before being placed in infusion bags. In some embodiments, TILs are cryopreserved without being placed in infusion bags. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is typically accomplished by placing a population of TILs in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are then placed in cryopreservation vials and stored at -80°C for 24 hours, with optional transfer to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some embodiments, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLifeSolutions). In some embodiments, the TIL population is cryopreserved using cryopreservation medium containing dimethyl sulfoxide (DMSO). In some embodiments, the TIL population is cryopreserved using a 1:1 (vol:vol) ratio of CS10 to cell culture medium. In some embodiments, the TIL population is cryopreserved using approximately a 1:1 (vol:vol) ratio of CS10 to cell culture medium (also containing additional IL-2).

As discussed above and illustrated in steps A through E in Figures 1 and/or 8 (especially, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P), cryopreservation can occur at multiple points in the TIL amplification process. In some embodiments, the expanded TIL population after the first amplification (as provided, for example, according to step B) or after one or more second amplifications according to step D of Figure 1 or Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be cryopreserved. Cryopreservation is typically accomplished by placing the TIL population in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are placed in cryopreservation vials and stored at -80°C for 24 hours, wherein optionally they are transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, TILs are cryopreserved according to the method provided in Example 6.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some cases, the TIL populations of step B in Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be immediately cryopreserved using the schemes discussed below. Alternatively, the main TIL population may undergo steps C and D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), and then cryopreservation following step D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Similarly, in cases where genetically modified TILs will be used in a therapy, the TIL population from step B or step D of Figure 1 or Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) may undergo genetic modification for appropriate treatment.

F. Phenotypic characteristics of amplified TILs

In some embodiments, the expression of TILs after amplification is analyzed using multiple phenotypic markers, including those described herein and in the examples. In some embodiments, the expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characterization of TILs is analyzed after the first amplification in step B of FIG1 or FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transition in step C from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transformation from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and after cryopreservation. In some implementations, the phenotypic characteristics of TIL are analyzed after the second amplification in step D from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed after two or more amplifications from step D of FIG1 or FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P).

In some implementations, the marker is selected from the group consisting of CD8 and CD28. In some implementations, the expression of CD8 is examined. In some implementations, the expression of CD28 is examined. In some implementations, compared to other processes (e.g., the Gen 3 process provided in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B, the expression of CD28 is examined. The 2A process provided in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) produces a higher expression of CD8 and/or CD28 on the TIL according to the process of the present invention. In some implementations, the expression of CD8 on the TIL produced by the process according to the invention is higher than that of other processes (e.g., the Gen 3 process provided in, for example, FIG8 (especially, for example, FIG8B)) and the 2A process provided in, for example, FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, the expression of CD28 on the TIL produced by the process according to the invention is higher than that of the 2A process provided, such as, for example, in Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to other processes (e.g., Figure 8A and/or Figure 2A, for example, Figure 8A). In some embodiments, high CD28 expression indicates a younger and more durable TIL phenotype. In some embodiments, the expression of one or more regulatory markers is measured.

In some implementations, during any step of the method for amplifying tumor-infiltrating lymphocytes (TILs) as described herein, no selection of a first TIL population, a second TIL population, a third TIL population, or a collected TIL population is made based on CD8 and/or CD28 expression.

In some embodiments, the percentage of central memory cells in the TIL produced by the method according to the invention is higher than that of process 2A, as provided in, for example, Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the memory markers of the central memory cells are selected from the group consisting of CCR7 and CD62L.

In some implementations, the CD4+ and/or CD8+ TIL memory subsets may be divided into different memory subsets. In some implementations, the CD4+ and/or CD8+ TILs contain initial (CD45RA+CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain central memory (CM; CD45RA-CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain effect memory (EM; CD45RA-CD62L-) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain RA+ effect memory/effect (TEMRA/TEFF; CD45RA+CD62L+) TILs.

In some embodiments, the TIL expresses one or more markers selected from the group consisting of granzyme B, perforin, and granulosin. In some embodiments, the TIL expresses granzyme B. In some embodiments, the TIL expresses perforin. In some embodiments, the TIL expresses granulosin.

In some embodiments, a cytokine release assay may also be used to evaluate cytokine release from restimulated TILs. In some embodiments, interferon-γ (IFN-γ) secretion from TILs may be evaluated. In some embodiments, IFN-γ secretion is measured by an ELISA assay. In some embodiments, IFN-γ secretion is measured by an ELISA assay after a rapid second amplification step, following step D as provided, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL health is measured by IFN-γ (IFN-γ) secretion. In some embodiments, IFN-γ secretion indicates active TILs. In some embodiments, a potency assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the culture medium of TILs stimulated with antibodies against CD3, CD28, and CD137/4-1BB. The IFN-γ level in the culture medium from these stimulated TILs can be determined by measuring IFN-γ release. In some embodiments, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), step D in the Gen 3 process provided in the TIL shows increased IFN-γ production compared to step D in the 2A process provided, for example, Figure 8 (especially, for example, Figure 8A), indicating an increased cytotoxic potential of the TIL in step D. In some embodiments, IFN-γ secretion is increased by one, two, three, four, or five times or more. In some embodiments, IFN-γ secretion is increased by one time. In some embodiments, IFN-γ secretion is increased by two times. In some embodiments, IFN-γ secretion is increased by three times. In some embodiments, IFN-γ secretion is increased by four times. In some embodiments, IFN-γ secretion is increased by five times. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ in ex vivo TILs is measured. In some embodiments, IFN-γ in ex vivo TILs is measured, including TILs produced by the methods of the present invention (including, for example, the method of Figure 8B).

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL to about 1000 pg/mL or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some implementations, it is capable of secreting at least 200 pg/mL, at least 250 pg/mL, at least 300 pg/mL, at least 350 pg/mL, at least 400 pg/mL, at least 450 pg/mL, at least 500 pg/mL, at least 550 pg/mL, at least 600 pg/mL, at least 650 pg/mL, at least 700 pg/mL, at least 750 pg/mL, at least 800 pg/mL, at least 850 pg/mL. L, at least 900 pg/mL, at least 950 pg/mL, or at least 1000 pg/mL or more of IFN-γ TILs are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL/5e5 cells to about 1000 pg/mL/5e5 cells or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, it is capable of secreting at least 200 pg/mL/5e5 cells, at least 250 pg/mL/5e5 cells, at least 300 pg/mL/5e5 cells, at least 350 pg/mL/5e5 cells, at least 400 pg/mL/5e5 cells, at least 450 pg/mL/5e5 cells, at least 500 pg/mL/5e5 cells, at least 550 pg/mL/5e5 cells, at least 600 pg/mL/5e5 cells, at least 650 pg/mL/5e5 cells, at least 700 pg/mL/5e5 cells, and at least 750 pg/mL/5e5 cells. TILs of IFN-γ produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) are TILs produced by at least 800 pg/mL/5e5 cells, at least 850 pg/mL/5e5 cells, at least 900 pg/mL/5e5 cells, at least 950 pg/mL/5e5 cells, or at least 1000 pg/mL/5e5 cells or more. In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that express and increase the diversity of T cell reservoirs. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method called Gen2 exemplified in Figure 8 (particularly, for example, Figure 8A). In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased. In some embodiments, processes as described herein (e.g., the Gen 3 process) exhibit higher clonal diversity based on the number of unique peptide CDRs within the sample compared to other processes (e.g., the Gen 2 process).

In some embodiments, TIL activation and depletion can be determined by examining one or more biomarkers. In some embodiments, activation and depletion can be determined using multicolor flow cytometry. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: CD3, PD-1, 2B4/CD244, CD8, CD25, BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183, CD69, CD95, CD127, CD103, and/or LAG-3. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, PD-1, TIGIT, and/or TIM-3. In some embodiments, activation and depletion of markers include (but are not limited to) one or more markers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, CD103+/CD69+, CD103+/CD69-, PD-1, TIGIT, and/or TIM-3. In some embodiments, T cell markers (including activation and depletion markers) may be identified and/or analyzed to examine T cell activation, suppression, or function. In some embodiments, T cell markers may include (but are not limited to) one or more markers selected from the group consisting of: TIGIT, CD3, FoxP3, Tim-3, PD-1, CD103, CTLA-4, LAG-3, BTLA-4, ICOS, Ki67, CD8, CD25, CD45, CD4, and/or CD59.

In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, TILs exhibiting secretion of granzyme B at levels higher than 1000 pg/mL to 300000 pg/mL or more are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed at levels above 1000 pg/mL, above 2000 pg/mL, above 3000 pg/mL, above 4000 pg/mL, above 5000 pg/mL, above 6000 pg/mL, above 7000 pg/mL, above 8000 pg/mL, above 9000 pg/mL, above 10000 pg/mL, above 20000 pg/mL, above 30000 pg/mL, above 40000 pg/mL, above 50000 pg/mL, and above 60000 pg/mL. TILs with granzyme B concentrations of 0000 pg/mL, above 70000 pg/mL, above 80000 pg/mL, above 90000 pg/mL, above 100000 pg/mL, or more are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 1000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 2000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 3000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 4000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 5000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 6000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 7000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 8000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 9000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 10,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 20,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 30,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 40,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 50,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 60,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 70,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 80,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 90,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 100,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of granzyme B above 260,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, the amplification method of the present invention produces an amplified TIL population exhibiting increased in vitro granzyme B secretion compared to a non-amplified TIL population, including, for example, the TILs provided in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased by at least one to fifty times or more compared to a non-amplified TIL population. In some embodiments, IFN-γ secretion is increased by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least thirty, at least forty, at least fifty times or more compared to a non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least doubled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least quadrupled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least five times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least six times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least seven times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least eight times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least nine-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least ten-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least twenty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least thirty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least forty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least fifty-fold compared to the non-amplified TIL population.

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times lower levels of TNF-α (i.e., TNF-alpha) compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α (i.e., TNF-alpha) are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, IFN-γ and granzyme B levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, granzyme B and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ, granzyme B, and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some implementations, phenotypic characteristics are examined after cryopreservation.

G. Other process implementation plans

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days or about 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days or about 1 to 10 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling-up of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein in the second container, the second TIL population from the small-scale culture is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days or approximately 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring and distributing the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 to 7 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for approximately 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for approximately 1 to 8 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a vertical scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 5 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a longitudinal scaling up of the culture by: (1) performing rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-g500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by contacting a first TIL population with a culture medium that also contains exogenous antigen-presenting cells (APCs), wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the culture medium is supplemented with additional exogenous APC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 2:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, or 2.3:1. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the initial first amplification is exactly or approximately 1× ^10⁸ , ^1.1 ×10⁸, 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, 1.5× ^10⁸ , 1.6 ^{×10⁸ ,} 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9×10⁸, 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , 3.1× ^10⁸ , or 3.1×10⁸. ⁸ , 3.2× ^10⁸ , 3.3× ^10⁸ , 3.4× ^10⁸ , or 3.5× ^10⁸ APCs, wherein the number of APCs added in the rapid second amplification is exactly or approximately 3.5× ^10⁸ , 3.6×10⁸, ^3.7 × ^10⁸ , 3.8× ^10⁸ , 3.9× ^10⁸ , 4×10⁸, 4.1× ^10⁸ , ^4.2 × ^10⁸ , 4.3× ^10⁸ , 4.4× ^10⁸ , 4.5×10⁸, 4.6× ^10⁸ , 4.7× ^10⁸ , 4.8× ^10⁸ , 4.9× ^10⁸ , 5× ^10⁸ , 5.1× ^10⁸ , 5.2× ^{10⁸ ,} or 5.3× ^10⁸. 5.4× ^10⁸ , 5.5× ^10⁸ , 5.6×10⁸, 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2×10⁸, 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , ^6.6 × ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , ^7.1 × ^10⁸ , 7.2×10⁸, 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , ^7.8 × ^10⁸ 7.9× ^10⁸ , 8× ^10⁸ , 8.1×10⁸, 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7×10⁸, 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , ^9.6 × ^10⁸ , 9.7× ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or} 1× ^10⁹ APCs.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1 × ^10⁸ APCs to exactly or about 3.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 3.5 × ^10⁸ APCs to exactly or about 1 × ^10⁹ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 2 × ^10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or approximately 2.5 × ^10⁸ APCs are added to an initial first amplification, and exactly or approximately 5 × ^10⁸ APCs are added to a rapid second amplification.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein multiple tumor fragments are distributed into multiple separate containers, in each separate container a first TIL population is obtained in step (a), a second TIL population is obtained in step (b), and a third TIL population is obtained in step (c), and therapeutic TIL populations from the multiple containers in step (c) are combined to produce a collected TIL population from step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of tumors are uniformly distributed into a plurality of separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise at least two separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to twenty separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to fifteen separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to ten separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to five separate containers.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the plurality of separate containers comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first amplification of a first TIL population in step (b) is performed in each container, and a rapid second amplification of a second TIL population generated from such a first TIL population is performed in the same container as in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each of the separate containers includes a first breathable surface area.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein multiple tumor fragments are distributed in a single container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a single container includes a first breathable surface region.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in a second container containing a second breathable surface region.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second container is larger than the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the second breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:9.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:2.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected as exactly or greater. Approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4... 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 1.5:1 to exactly or about 100:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 50:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 25:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 50 times larger in number than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times larger than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is supplemented with additional IL-2 exactly or about 2 days or exactly or about 3 days after the start of the second time period in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, further comprising the step of cryopreserving the collected TIL population in step (d) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, including an additional step (e) after step (d) of transferring the collected TIL population from step (d) to an infusion bag optionally containing HypoThermosol.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, comprising the step of cryopreserving an infusion bag containing the collected TIL population in step (e) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation process is performed using a 1:1 ratio of collected TIL populations to cryopreservation medium.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (b) is 2.5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (c) is 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a PBMC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are artificial antigen-presenting cells.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a membrane-based cell processing system is used for the collection in step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the collection in step (d) is performed using the LOVO cell processing system.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 5 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 10 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 15 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 20 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 25 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 30 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 35 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 40 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 45 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or about 50 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 segments per container.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately 27 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 20 ^mm³ to exactly or about 50 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 21 ^mm³ to exactly or about 30 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 22 ^mm³ to exactly or about 29.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 23 ^mm³ to exactly or about 29 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 24 ^mm³ to exactly or about 28.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 25 ^mm³ to exactly or about 28 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 26.5 ^mm³ to exactly or about 27.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately the following: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 30 to exactly or about 60 segments, wherein the total volume is exactly or about 1300 ^mm³ to exactly or about 1500 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total volume is exactly or about 1350 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total mass is exactly or about 1 gram to exactly or about 1.5 grams.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is provided in a container in the form of a G container or a Xuri cell bag.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is from about 10,000 IU/mL to about 5,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is about 6,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second time period in step (c) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 5 days, 6 days, or 7 days, respectively.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 16 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 17 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 16 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 14 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 15 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 14 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 15 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 14 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 15 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 16 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 18 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the therapeutic TIL population collected in step (d) contains a therapeutically effective dose of TIL sufficient for use with TIL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of TILs sufficient for a therapeutically effective dose is exactly or about 2.3 × 10¹⁰ to exactly or about 13.7 × 10¹⁰.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein effector T cells and/or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression relative to effector T cells and/or central memory T cells obtained from the second cell population in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a closed container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a G container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-100.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-500.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared by the methods described in any of the foregoing paragraphs as applicable above.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs) or OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of antigen-presenting cells (APCs) and OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 16 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 17 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 18 days.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased interferon-γ production.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased polyclonalness.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased efficacy.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A through F above, or according to steps A through F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than one times the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than twice the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least three times more interferon-γ.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any antigen-presenting cells (APCs). In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are made capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that is capable of producing at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are able to produce at least one times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least three times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy), a core needle biopsy, a core needle puncture biopsy, or a fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a fine-needle aspirate.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is obtained by core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirates; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first amplification step; (iii) the method includes a period of approximately 8 days for initiating the first amplification; and (iv) the method includes a period of approximately 11 days for rapid second amplification. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating the first expansion step; (iii) the method includes a period of approximately 8 days for initiating the first expansion; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing the subcultures for approximately 6 days. In some of the foregoing embodiments, up to 5 subcultures are cultured separately in containers of the same size or larger as the container in which the second TIL population is initially cultured in the rapid second expansion. In some of the foregoing embodiments, the culture of the second TIL population is evenly distributed among up to 5 subcultures. In some of the aforementioned embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1 to about 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step to obtain a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days; and (iv) the method includes a rapid second expansion step by culturing the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 11 days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days to obtain a second TIL population; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing each of the subcultures in a cell culture medium containing IL-2 for approximately 6 days. In some of the foregoing embodiments, up to five subcultures are cultured in containers of the same size or larger than the container in which the second TIL population was started during the rapid second amplification. In some of the foregoing embodiments, the cultures of the second TIL population are evenly distributed among up to five subcultures. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method comprises obtaining a first TIL population from 1 to about 10 tumor tissue samples from a subject by core needle biopsy; (ii) the method comprises, prior to performing the first amplification step, culturing the first TIL population in a G-REX-100M culture flask in a cell culture medium containing 0.5 L CM1 medium of 6000 IU IL-2/mL for about 3 days; (iii) the method comprises initiating the first amplification by adding 0.5 L CM1 medium containing 6000 IU/mL IL-2, 30 ng/mL OKT-3, and about ^10⁸ feeder cells, and culturing for about 8 days; and (iv) the method comprises performing a rapid second amplification by transferring the second TIL population to a 5 L CM1 medium containing 3000 IU/mL IL-2, 30 ng/mL OKT-3, and 5 × ^10⁹ feeder cells. (a) The culture is divided into up to five subcultures by transferring ^10⁹ TILs into each of up to five G-REX-500MCS culture flasks containing 5 L AIM-V medium with 3000 IU/mL IL-2, and said subcultures are cultured for approximately six days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (a) initiating a first expansion of a first T cell population obtained from a donor by culturing a first T cell population to achieve growth and initiate activation of the first T cell population; (b) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (c) collecting the second T cell population. In other embodiments, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling up of culture scale by: (a) rapidly expanding the first T cell population by culturing it in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second expansion step is broken down into multiple steps to achieve a longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of up to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), the first T cell population is cultured in a first culture medium containing OKT-3 and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises a 4-1BB agonist, OKT-3, and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the first T cell population is cultured in a second culture medium containing OKT-3, IL-2 and antigen-presenting cells (APC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains 4-1BB agonist, OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs in the second APC group to the number of APCs in the first APC group is about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs in the first APC group is about 2.5 × ^10⁸ and the number of APCs in the second APC group is about 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is laminated onto a first breathable surface with an average thickness of two APC layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC group is laminated onto the first breathable surface with an average thickness selected from 4 to 8 APC layers.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the average number of APC layers stacked on the first breathable surface in step (b) to the average number of APC layers stacked on the first breathable surface in step (a) is 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 2.0 × ^10⁶ APCs/ ^cm² to just or about 3.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 2.5 × ^10⁶ APCs/ ^cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 2.0 × ^10⁶ APCs/ ^cm² to exactly or about 3.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a peripheral blood mononuclear cell (PBMC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMCs are irradiated and the donor for the first T cell population is exogenous.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are bone marrow infiltrating lymphocytes (MIL).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are peripheral blood lymphocytes (PBLs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by separation from whole blood of a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by isolating from the donor's apheresis product.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first T cell population is isolated from whole blood or apheresis products of a donor by positive or negative selection of T cell phenotype.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cell phenotype is CD3+ and CD45+.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein T cells are isolated from NK cells prior to initiating a first expansion of a first T cell population. In other embodiments, T cells in a first T cell population are separated from NK cells by removing CD3-CD56+ cells from the first T cell population. In other embodiments, CD3-CD56+ cells are removed from the first T cell population by using a gating strategy that removes CD3-CD56+ cell fractions and recovers negative fractions. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of NK cells. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of CD3-CD56+ cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of NK cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of NK cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with ovarian cancer.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or about 1 × ^10⁷ T cells from a first T cell population are seeded into a container to initiate an initial first expansion culture in such a container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein a first T cell population is distributed into a plurality of containers, and exactly or about 1 × 10⁷ T cells from the first T cell population are seeded in each container to initiate an initial first expansion culture in such containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second T cell population collected in step (c) is a therapeutic TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via small biopsy (including, for example, perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a donor, including small biopsies (such as perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsy of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a donor.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsies, core needle biopsies, or needle biopsies obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the initiation of the first expansion is performed in a container containing a first breathable surface region, wherein the initiation of the first expansion is performed for a first time period of approximately 7 or 8 days to obtain the second TIL population. (iii) A second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iv) a rapid second expansion is performed by supplementing the second TIL population with a second cell culture medium containing additional IL-2, OKT-3 and APC to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (ii), wherein the rapid second expansion is performed for a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container containing a second ventilated surface area; (v) the therapeutic TIL population obtained from step (iii) is collected; and (v) the collected TIL population from step (iv) is transferred to an infusion bag.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsy, core needle biopsy, or needle biopsy samples obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; and (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a second TIL population, wherein the initiation... The first amplification is carried out over a first period of approximately 7 or 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iii) a rapid second amplification is carried out by contacting the second TIL population with a third cell culture medium containing IL-2, OKT-3 and APC to produce a third TIL population, wherein the rapid second amplification is carried out over a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second amplification is carried out in a container containing a second ventilated surface area; and (iv) the therapeutic TIL population obtained from step (iii) is collected.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with an additional amount of third culture medium and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with a fourth medium containing IL-2 and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the culture is split into up to five subcultures after day 5 of the second time period.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein all steps in the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (i) initiating a first expansion of a first T cell population by culturing a first T cell population to achieve growth and initiate activation of the first T cell population, the first T cell population being derived from a tumor sample obtained from one or more small biopsies, core needle biopsies, or needle biopsies of a tumor in a donor; (ii) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population a second T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (iv) collecting the second T cell population. In some embodiments, the tumor sample is obtained from multiple core needle biopsies. In some embodiments, the multiple core needle biopsies are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, and 10 core needle biopsies.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein T cells or TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubating the tumor in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, California). In some embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes one or more dissociation (digestive) enzymes, such as (but not limited to) collagenase (including any blended or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersant enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, protease type XIV (streptomycin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof. In other embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes collagenase (including any blended or type of collagenase), neutral protease (dispersant enzyme), and deoxyribonuclease I (DNase).

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a TIL population undergoes a gene-editing process. In some embodiments, the gene-editing process may be performed at any time during the TIL amplification process, including before, during, or after any step of the amplification process, such as before, during, or after one or more of steps A, B, C, D, E, and F outlined above.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein an activation step follows a first amplification step. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and/or anti-CD38 beads for about 1-7 days. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and anti-CD38 beads for about 1-7 days. In some embodiments, the activation step is performed for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the activation step is followed by a gene editing step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a gene editing step follows a first amplification step. In some embodiments, when the first amplification step includes OKT-3, a gene editing step follows the first amplification step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the gene editing step includes a sterile electroporation step on a TIL population. In some embodiments, the sterile electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates the expression of PD-1. According to some embodiments, the gene editor further comprises a TALE nuclease system that downregulates the expression of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CTLA-4 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/LAG-3 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CISH double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CTLA-4. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and TIGIT. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and TIGIT. According to some implementation schemes, the obtained TILs showed downregulation of CTLA-4 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CISH expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of TIGIT and CBL-B expression.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a quiescent step follows the gene editing step. In some embodiments, the quiescent step comprises incubating a TIL population at about 30-40°C and about 5% _CO2 for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 30°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1 hour, followed by about 15-23 hours at about 30°C.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a second amplification step follows the resting step.

VIII. Pharmaceutical Compositions, Dosage, and Dosing Regimens

In some embodiments, TILs, MILs, or PBLs (including TILs, MILs, or PBLs genetically modified to express CCR) amplified and/or genetically modified using the methods of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC or melanoma. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is metastatic NSCLC or metastatic melanoma. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of NSCLC. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

In other embodiments, the present invention provides an infusion bag that includes the therapeutic TIL population described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a pharmaceutically acceptable carrier as described in any of the preceding paragraphs.

In other embodiments, the present invention provides an infusion bag comprising the TIL composition described in any of the preceding paragraphs.

In other embodiments, the present invention provides a cryopreservation formulation for a therapeutic TIL population as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a cryopreservation culture medium as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains DMSO.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a cryopreservation formulation of the TIL composition described in any of the preceding paragraphs.

In some embodiments, TILs amplified using the methods of this disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly when the cancer is NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

IX. Methods of treating patients

The treatment method begins with the collection and culture of raw TILs. Such methods have been described in the art, for example, by reference in their entirety to Jin et al., J. Immunotherapy, 2012, 35(3):283-292. The implementation of the treatment method is described throughout the following sections, including examples.

The methods described herein, including, for example, those described in steps A through F above or those described in steps A through F above (and also, for example, those shown in Figures 1, 36, and/or 8), have been found to have specific uses in the treatment of cancer patients (e.g., as described in Goff et al., J. Clinical Oncology, 2016, 34(20):2389-239 and the Supplement, which are incorporated herein by reference in their entirety). In some embodiments, TILs are grown from resected metastatic melanoma remnants as previously described (see Dudley et al., J Immunother., 2003, 26:332-342, which are incorporated herein by reference in their entirety). Fresh tumors can be segmented under sterile conditions. Representative samples can be collected for formal pathological analysis. Single fragments of 2 ^mm³ to ³ mm³ can be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained from each patient. In some embodiments, 24 samples are obtained from each patient. Samples can be placed in individual wells of a 24-well plate, maintained in growth medium containing a high dose of IL-2 (6,000 IU/mL), and tumor destruction and/or TIL proliferation can be monitored. As described herein, any tumor cells remaining viable after treatment can be enzymatically digested into a single-cell suspension and cryopreserved.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumors when available. TILs are considered reactive if overnight co-culture produces interferon-γ (IFN-γ) levels >200 pg/mL and twice the background level. (Goff et al., J Immunother., 2010, 33:840-847; incorporated herein by reference in its entirety). In some embodiments, cultures demonstrating autoreactivity or adequate growth patterns may be selected for secondary amplification (e.g., the secondary amplification provided according to step D of Figures 1, 36, and/or 8), including what is sometimes referred to as rapid amplification (REP). In some embodiments, amplified TILs exhibiting high autoreactivity (e.g., high proliferation during secondary amplification) are selected for additional secondary amplification. In some implementations, TILs with high self-reactivity (e.g., high proliferation during the second amplification provided in step D of Figures 1, 36 and/or 8) are selected for additional second amplification according to step D of Figures 1, 36 and/or 8.

Cell phenotypes of cryopreserved samples of infusion bag TILs can be analyzed by flow cytometry targeting surface markers CD3, CD4, CD8, CCR7, and CD45RA (e.g., FlowJo) (BD BioSciences) and by any of the methods described herein. Serum cytokines were measured using standard enzyme-linked immunosorbent assay (ELISA). An increase in serum IFN-γ was defined as a baseline level >100 pg/mL and greater than 43.

In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, achieve remarkable improvements in clinical efficacy. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, exhibit improved clinical efficacy compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8). In some embodiments, methods other than those described herein include methods referred to as Process 1C and/or Gen 1. In some embodiments, the increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1 or 36, exhibit similar response time and safety profiles compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8).

In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of a subject treated with TIL indicates active TIL. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TIL of a subject treated with TIL prepared by the methods of the present invention, including, for example, those described in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TIL produced by the methods of the present invention. In some embodiments, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or compared to patients treated with TIL prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is quadrupled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is increased fivefold compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the serum of subjects treated with TILs prepared using the methods of the present invention, including those prepared, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy in treating tumors.

In some embodiments, the TILs prepared by the methods of the present invention include those described, for example, in Figure 1 or Figure 36, where IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of subjects treated with TILs indicates active TILs. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TILs of subjects treated with TILs prepared by the methods of the present invention, including methods described, for example, in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TILs produced by the methods of the present invention. In some implementations, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, and/or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, and/or 8, such as those referred to as the process 1C method). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, those other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

Measures of efficacy may include disease control rate (DCR) and overall response rate (ORR), as known in the art and described herein.

A. Methods of treating cancer

The compositions and methods described herein can be used in a method of treating a disease. In some embodiments, they are used to treat hyperproliferative conditions, such as cancer, in adult or pediatric patients. They can also be used to treat other conditions as described herein and in the following paragraphs.

In some implementations, hyperplasia is defined as cancer. In some implementations, hyperplasia is defined as solid tumor cancer. In some implementations, solid tumor cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papillomavirus (HPV), central nervous system-related cancers (including ependymoma, neuroblastoma, neuroblastoma, pineal blastoma, and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSC)). C) Hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, and pharyngeal cancer; kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC), metastatic NSCLC, and small cell lung cancer); melanoma (including uveal melanoma, choroidal melanoma, ciliary body melanoma, iris melanoma, or metastatic melanoma); mesothelioma (including malignant pleural mesothelioma); ovarian cancer; pancreatic cancer (including pancreatic duct adenocarcinoma); penile cancer; rectal cancer; kidney cancer; renal cell carcinoma; sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas); thyroid cancer (including anaplastic thyroid cancer); uterine cancer and vaginal cancer.

In some embodiments, the hyperplastic condition is a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from the group consisting of: chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-B, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified MIL or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-BR, wherein the cancer is a hematologic malignancy.

In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, that is relapsed or refractory to treatment with at least one prior therapy (including chemotherapy, radiotherapy, or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least two prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least three prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy).

In some embodiments, the cancer is a microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. MSI-H and dMMR cancers and their detection have been described in Kawakami et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosure of which is incorporated herein by reference.

In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a non-human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a companion animal.

In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors and/or MEK inhibitors. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method of treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, enrafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof; and difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, bemetinib, selmetinib, pimatetinib, refatinib, and pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is a pediatric cancer.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is uveal melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the uveal melanoma is a choroidal melanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is neuroblastoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a central nervous system (CNS) related cancer. In some embodiments, the pediatric cancer is difficult to treat with chemotherapy. In some embodiments, the pediatric cancer is difficult to treat with radiation therapy. In some embodiments, the pediatric cancer is difficult to treat with dinutuximab.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the CNS-related cancer is neuroblastoma, pinealoblastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein can be used to treat cancer that is refractory to anti-PD-1 or anti-PD-L1 antibodies or resistant to prior treatment. In some embodiments, the patient is a primary refractory patient to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has not shown a prior response to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has shown a prior response to anti-PD-1 or anti-PD-L1 antibodies, followed by cancer progression. In some embodiments, the cancer is refractory to combination therapy with anti-CTLA-4 antibodies and/or anti-PD-1 or anti-PD-L1 antibodies in combination with at least one chemotherapeutic agent. In some embodiments, the prior chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and/or cisplatin. In some prior embodiments, the chemotherapeutic agent is a platinum-based dual chemotherapeutic agent. In some embodiments, the platinum dual therapy comprises a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel, docetaxel, or nab-paclitaxel). In some embodiments, the platinum dual chemotherapeutic agent is combined with pemetrexed.

In some implementations, NSCLC is PD-L1 negative and/or comes from patients with cancers that express PD-L1 and have a tumor proportion score (TPS) <1%, as described elsewhere in this article.

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or anti-PD-L1 antibody, pemetrexed, and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some embodiments, NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient has not received treatment. In some embodiments, NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, NSCLC has been previously treated with a chemotherapy agent. In some embodiments, NSCLC has been previously treated with a chemotherapy agent but is no longer being treated with said chemotherapy agent. In some embodiments, the NSCLC patient has not received anti-PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the NSCLC patient has not received NSCLC treatment or has received chemotherapy agent treatment but has not received PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has not received treatment or has received chemotherapy agent treatment but has not received anti-PD-1/PD-L1 treatment and has low PD-L1 expression. In some embodiments, the NSCLC patient had massive disease at baseline. In some embodiments, the subject has massive disease and low PD-L1 expression at baseline. In some embodiments, the NSCLC patient does not have detectable PD-L1 expression. In some embodiments, the NSCLC patient is untreated or has received chemotherapy but not anti-PD-1/PD-L1 treatment and does not have detectable PD-L1 expression. In some embodiments, the patient has massive disease and does not have detectable PD-L1 expression at baseline. In some embodiments, the NSCLC patient has untreated NSCLC or has received chemotherapy (e.g., received chemotherapy agents) but not anti-PD-1/PD-L1 treatment, and the patient has low PD-L1 expression and/or has massive disease at baseline. In some embodiments, massive disease is indicated when the maximum tumor diameter measured in the transverse or coronal plane is greater than 7 cm. In some embodiments, massive disease is indicated when there are swollen lymph nodes with a short axis diameter of 20 mm or greater. In some embodiments, the chemotherapy agents include standard of care treatments for NSCLC.

In some embodiments, PD-L1 expression is determined by a tumor proportion score. In some embodiments, a subject with refractory NSCLC has a tumor proportion score (TPS) of <1%. In some embodiments, a subject with refractory NSCLC has a TPS of ≥1%. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment.

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, or 8, including, for example, methods referred to as Process 1C). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy in cancer treatment. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8).

In some embodiments, PD-L1 expression is determined by tumor proportion scoring using one or more assays as described herein. In some embodiments, the subject or patient with NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the NSCLC tumors have a TPS of ≥1%. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a TPS of ≥1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and a tumor proportion score has been determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment.

In some implementations, NSCLC is NSCLC exhibiting a tumor proportion score (TPS), or the percentage of live tumor cells obtained from the patient prior to anti-PD-1 or anti-PD-L1 therapy, or the percentage of PD-L1 protein showing partial or complete membrane staining at any intensity of less than 1% (TPS<1%). In some implementations, NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, <0.04%, <0.03%, <0.02%, and <0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, and about 0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS between 0% and 1%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.9%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.8%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.7%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.6%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.5%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.4%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.3%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.2%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.1%. TPS can be measured by methods known in the art, such as those described in Hirsch et al., J. Thorac. Oncol. 2017, 12, 208-222, or methods used to determine TPS prior to treatment with pembrolizumab or other anti-PD-1 or anti-PD-L1 therapies. Methods approved by the U.S. Food and Drug Administration for measuring TPS can also be used. In some embodiments, PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

In some embodiments, partial membrane staining includes 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more. In some embodiments, complete membrane staining includes approximately 100% membrane staining.

In some implementations, testing PD-L1 may involve measuring the level of PD-L1 in a patient's serum. In these implementations, measuring PD-L1 in a patient's serum eliminates the uncertainty of tumor heterogeneity and the discomfort of patients undergoing serial biopsies.

In some implementations, elevated soluble PD-L1 levels compared to baseline or standard levels are associated with a prognostic risk of NSCLC progression. See, for example, Okuma et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli et al., Oncotarget, 2018, 9, 17554-17563. In some implementations, PD-L1 is exosomal PD-L1. In some implementations, PD-L1 is expressed on circulating tumor cells.

In some implementation schemes, the subject or patient has non-small cell lung cancer (NSCLC), characterized by at least one of the following:

i. PD-L1 pre-defined tumor proportion score (TPS) <1%,

ii. The TPS score of PD-L1 is 1%-49%, or

iii. The predetermined absence of one or more driver mutations,

The driving mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20 mutations, KRAS mutations, BRAF mutations, ALK mutations, c-ROS mutations (ROS1 mutations), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and/or altered MET signals, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations, and GNA11 mutations.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population described herein.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition described herein.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to the administration of therapeutically effective doses of the therapeutic TIL population and TIL composition described herein, respectively.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, the method further comprising the step of initiating treatment of the subject with a high-dose IL-2 regimen the day after administration of TIL cells to the subject.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg every eight hours via a 15-minute bolus intravenous infusion until tolerated.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is glioblastoma (including GBM).

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides a therapeutic TIL population as described herein in a method for treating a subject suffering from cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer using a TIL composition described herein, comprising administering a therapeutically effective dose of the TIL composition to the subject.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administration of a therapeutically effective dose of the therapeutic TIL population or TIL composition described herein.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, which further includes the step of starting treatment of the patient with a high-dose IL-2 regimen the day after administration of TIL cells.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg via intravenous infusion every eight hours in the form of 15-minute bolus injections until tolerated.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is glioblastoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides the use of the therapeutic TIL population described herein in a method of treating a subject’s cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides the use of the TIL composition described in any of the foregoing paragraphs in a method of treating a subject’s cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In other embodiments, the present invention provides the use of the therapeutic TIL population or the TIL composition described herein in a method of treating a patient with cancer, the method comprising administering a non-myeloablative lymphocyte depletion regimen to the patient and then administering to the subject a therapeutically effective dose of the therapeutic TIL population or the therapeutically effective dose of the TIL composition described herein in any of the preceding paragraphs.

1. Combination with PD-1 and PD-L1 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, which includes TIL and one or more PD-1 and/or PD-L1 inhibitors.

Programmed death 1 (PD-1) is a 288-amino acid transmembrane immune checkpoint receptor protein expressed by T cells, B cells, natural killer (NK) T cells, activated monocytes, and dendritic cells. PD-1, also known as CD279, belongs to the CD28 family and is encoded by the Pdcd1 gene on chromosome 2 in humans. PD-1 consists of an immunoglobulin (Ig) superfamily domain, a transmembrane domain, and an intracellular domain containing the immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoreceptor tyrosine switching motif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play important roles in immune tolerance, as described by Keir et al., Annu. Rev. Immunol. 2008, 26, 677-704. PD-1 provides a suppressive signal that negatively regulates T cell immune responses. PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273) are expressed on tumor cells and stromal cells, which may encounter activated T cells expressing PD-1, leading to immunosuppression of T cells. PD-L1 is a 290-amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9. Blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2 using PD-1 inhibitors, PD-L1 inhibitors, and/or PD-L2 inhibitors can overcome immune resistance, as demonstrated in recent clinical studies, such as those described in Topalian et al., N. Eng. J. Med. 2012, 366, 2443-54. PD-L1 is expressed on many tumor cell lines, while PD-L2 is primarily expressed on dendritic cells and some tumor lines. In addition to T cells (which induce PD-1 expression upon activation), PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes, and dendritic cells.

In some embodiments, the PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail below. Regarding PD-1 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, references herein to PD-1 inhibitors as antibodies may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, references herein to PD-1 inhibitors may also refer to small molecule compounds or their pharmaceutically acceptable salts, esters, solvates, hydrates, cocrystals, or prodrugs.

In some embodiments, the PD-1 inhibitor is an antibody (i.e., an anti-PD-1 antibody) or a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In some embodiments, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competitively binds to PD-1 and/or binds to an epitope on PD-1. In some embodiments, the antibody competitively binds to PD-1 and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a KD of about 100 pM or less, at a KD of about 90 pM or less, at a KD of about 80 pM or less, at a KD of about 70 pM or less, at a KD of about 60 pM or less, at a KD of about 50 pM or less, at a KD of about 40 pM or less, at a KD of about 30 pM or less, at a KD of about 20 pM or less, at a KD of about 10 pM or less, or at a KD of about 1 pM or less.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a k _{-association rate} of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about ⁸ × 10⁵ l/M·s or faster, at a k _{-association rate} of about 8.5 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9.5 × ^10⁵ l/M·s or faster, or at a k- _{association rate} of about 1 × ^10⁶ l/M·s or faster.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, or with a k _{-dissociation rate} of about 2.1 × ^10⁻⁵ 1/s or slower, or with a k-dissociation rate of about 2.2 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.3 × 10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.6 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.7 × ^10⁻⁵ 1/s or slower. It binds to human PD-1 at a k- _{dissociation rate} of 1/s or slower, at a k- _{dissociation rate} of approximately 2.8 × ^10⁻⁵ 1/s or slower, at a k- _{dissociation rate} of approximately 2.9 × ^10⁻⁵ 1/s or slower, or at a k- _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, or about 6 nM or less. The binding of human PD-L2 to human PD-1; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower; or blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-1 inhibitor is nivolumab (available from Bristol-Myers Squibb via OPDIVO) or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-1 receptor. In some embodiments, the anti-PD-1 antibody is an immunoglobulin G4κ anti-(human CD274) antibody. Nivolumab is designated Chemical Abstracts Service (CAS) Registry No. 946414-94-4 and is also known as 5C4, BMS-936558, MDX-1106, and ONO-4538. The preparation and characteristics of nivolumab are described in U.S. Patent No. 8,008,449 and International Patent Publication No. WO 2006/121168, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of nivolumab in various forms of cancer have been described in Wang et al., Cancer Immunol. Res. 2014, 2, 846-56; Page et al., Ann. Rev. Med., 2014, 65, 185-202; and Weber et al., J. Clin. Oncology, 2013, 31, 4311-4318, the contents of which are incorporated herein by reference. The amino acid sequence of nivolumab is presented in Table 18. Nivolumab has intra-heavy chain disulfide bonds at 22-96, 140-196, 254-314, 360-418, 22”-96”, 140”-196”, 254”-314”, and 360”-418”; intra-heavy chain disulfide bonds at 23”-88”, 134”-194”’, 23”’-88”’, and 134”’-194”’; inter-heavy chain disulfide bonds at 127-214”’ and 127”-214”’; inter-heavy chain disulfide bonds at 219-219” and 222-222”; and N-glycosylation sites (H CH2 84.4) at 290” and 290”.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:158 and the light chain provided in SEQ ID NO:159. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO:159, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of nivolumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:160, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:161 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:165, SEQ ID NO:166, and SEQ ID NO:167, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of nivolumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab.

Table 18. Amino acid sequences of PD-1 inhibitors associated with nivolumab.

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at the following doses: about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 240 mg every 2 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 480 mg every 4 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma, with approximately 1 mg/kg of nivolumab administered every 3 weeks on the same day, followed by 3 mg/kg of ipilimumab for 4 consecutive doses, then 240 mg every 2 weeks or 480 mg of nivolumab every 4 weeks.

In some implementations, nivolumab is administered as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks and ipilimumab at approximately 1 mg/kg every 6 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks, plus ipilimumab at 1 mg/kg every 6 weeks and two cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks or 480 mg every 4 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks and ipilimumab at 1 mg/kg every 6 weeks to treat malignant pleural mesothelioma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks and 480 mg of nivolumab every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks to treat pediatric patients <40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 240 mg nivolumab every 2 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 480 mg nivolumab every 4 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab is administered every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 480 mg of nivolumab is administered every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor comprises pembrolizumab (available from Merck Inc. of Kenilworth, NJ, USA under the KEYTRUDA license) or its antigen-binding fragment, conjugate, or variant. Pembrolizumab is designated CAS Registry No. 1374853-91-4 and is also known as lanizumab, MK-3475, and SCH-900475. Pembrolizumab is an immunoglobulin G4 anti-(human protein PDCD1 (programmed cell death 1)) antibody containing a (human-mouse monoclonal heavy chain) disulfide bond and a human-mouse monoclonal light chain dimer structure. The structure of pembrolizumab can also be described as an immunoglobulin G4 anti-(human programmed cell death 1) antibody; containing a humanized mouse monoclonal [228-L-proline (H10-S>P)]γ4 heavy chain (134-218') disulfide bond and a humanized mouse monoclonal κ light chain dimer (226-226”:229-229”) disulfide bond. The properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO 2008/156712 A1, US Patent No. 8,354,509, and US Patent Application Publications Nos. US2010/0266617 A1, US2013/0108651A1, and US2013/0109843 A2, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of pembrolizumab in various forms of cancer are described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert et al., Lancet, 2014, 384, 1109-17; and Thomas et al., Exp. Opin. Biol. Ther., 2014, 14, 1061-1064. The amino acid sequence of pembrolizumab is illustrated in Table 19. Pembrolizumab includes the following disulfide bridges: 22-96, 22”-96”, 23”-92”’, 23”’-92”’, 134-218”, 134”-218”’, 138”-198”, 138”’-198”’, 147-203”, 147”-203”, 226-226”, 229-229”, 261-321”, 261”-321”, 367-425”, and 367”-425”; and the following glycosylation sites. (N): Asn-297 and Asn-297". Pembrolizumab is an IgG4/κ isoform containing a stable S228P mutation in the Fc region; the insertion of this mutation in the IgG4 hinge region prevents the formation of the half-molecule typically observed in IgG4 antibodies. Pembrolizumab is heteroglycosylated at Asn297 in the Fc domain of each heavy chain, resulting in a molecular weight of approximately 149 kDa for the complete antibody. The major glycoform of pembrolizumab is the fucosylated degalactosyl biantennary (GOF) form.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:168 and the light chain provided in SEQ ID NO:169. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of pembrolizumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:170, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:171 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises _{VH and VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises VH and VL regions} , each at least 97% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:175, SEQ ID NO:176, and SEQ ID NO:177, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of PD-1 approved by a regulatory agency, referencing pembrolizumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab.

Table 19. Amino acid sequences of PD-1 inhibitors associated with pembrolizumab.

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat melanoma. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat melanoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat melanoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat NSCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat small cell lung cancer (SCLC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat SCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat SCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat head and neck squamous cell carcinoma (HNSCC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat HNSCC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HNSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, children are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat MSI-H or dMMR cancer. In some implementations, pediatrics are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat MSI-H or dMMR cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient colorectal cancer (dMMR CRC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat MSI-H or dMMR CRC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat gastric cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat gastric cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat esophageal cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat esophageal cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cervical cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cervical cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat hepatocellular carcinoma (HCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are treated with pembrolizumab at approximately 200 mg every 3 weeks for Merkel cell carcinoma (MCC). In some implementations, adults are treated with pembrolizumab at approximately 400 mg every 6 weeks for MCC. In some implementations, children are treated with pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks for MCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat renal cell carcinoma (RCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks and axitinib 5 mg is administered orally twice daily to treat RCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat endometrial cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks, and lenvatinib 20 mg is administered orally once daily for non-MSI-H or dMMR tumors to treat endometrial cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are given pembrolizumab at approximately 200 mg every 3 weeks to treat TMB-H cancer. In some implementations, adults are given pembrolizumab at approximately 400 mg every 6 weeks to treat TMB-H cancer. In some implementations, children are given pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat TMB-H cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cutaneous squamous cell carcinoma (cSCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat triple-negative breast cancer (TNBC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat TNBC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, if the patient or subject is an adult, i.e., for the treatment of an adult indication, an additional dosing regimen of 400 mg every 6 weeks may be used. In some implementations, pembrolizumab administration may be initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration may be initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 clone J43 (catalog number BE0033-2) and RMP1-14 (catalog number BE0146) (Bio XCell, West Lebanon, New Hampshire, USA). Many commercially available anti-PD-1 antibodies are known to those skilled in the art.

In some embodiments, the PD-1 inhibitor is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publications Nos. 2010/0266617A1, 2013/0108651A1, and 2013/0109843A2, the disclosures of which are incorporated herein by reference. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody described in U.S. Patent Nos. 8,287,856, 8,580,247, and 8,168,757 and U.S. Patent Application Publications Nos. 2009/0028857A1, 2010/0285013A1, 2013/0022600A1, and 2011/0008369A1, the teachings of which are hereby incorporated by reference. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in U.S. Patent No. 8,735,553B1, the disclosure of which is incorporated herein by reference. In some embodiments, the PD-1 inhibitor is pilithumab, also known as CT-011, described in U.S. Patent No. 8,686,119, the disclosure of which is incorporated herein by reference.

In some embodiments, the PD-1 inhibitor may be a small molecule or a peptide or peptide derivative, such as those described in U.S. Patent Nos. 8,907,053, 9,096,642, and 9,044,442, and U.S. Patent Application Publication No. US2015/0087581; 1,2,4-oxadiazole compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2015/0073024; cyclic peptide mimics and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042; cyclic compounds and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042. Those described in US2015/0125491; 1,3,4-oxadiazole and 1,3,4-thiadiazole compounds and derivatives, such as those described in International Patent Application Publication WO 2015/033301; peptide-based compounds and derivatives, such as those described in International Patent Application Publications WO 2015/036927 and WO 2015/04490; or peptide-based macrocyclic compounds and derivatives, such as those described in US Patent Application Publication US2014/0294898; the disclosure of each of these patents is hereby incorporated by reference in its entirety. In some embodiments, the PD-1 inhibitor is cemiplimab, which is available from Regeneron Corporation.

In some embodiments, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors, antagonists, or blockers described in more detail below. Regarding PD-L1 and PD-L2 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor may also refer to the compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

In some embodiments, the compositions, processes, and methods described herein include PD-L1 or PD-L2 inhibitors. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2. In some embodiments, the antibody competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selective for PD-L1 because the concentration at which the compound binds to or interacts with PD-L1 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L2 receptor. In some embodiments, the compound binds to the PD-L1 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L2 receptor.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2 because the concentration at which the compound binds to or interacts with PD-L2 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L1 receptor. In some embodiments, the compound binds to the PD-L2 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L1 receptor.

Unbound by any theory, it is believed that tumor cells express PD-L1, and T cells express PD-1. However, tumor cell expression of PD-L1 is not required for the efficacy of PD-1 or PD-L1 inhibitors or blockers. In some embodiments, tumor cells express PD-L1. In other embodiments, tumor cells do not express PD-L1. In some embodiments, the method may include a combination of PD-1 and PD-L1 antibodies (e.g., the PD-1 and PD-L1 antibodies described herein) with TIL. The combination of PD-1 and PD-L1 antibodies with TIL may be administered simultaneously or sequentially.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that binds to human PD-L1 and/or PD-L2 with a KD of about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, or about 30 pM or less.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that _{bind to human PD-L1 and/or PD-L2 at a k-association rate of} about ^7.5 × 10⁵ ¹ /M·s or faster, at about 8 × 10⁵ 1/M·s or _faster , at about 8.5 × 10⁵ ¹ /M·s or faster, at about 9 × ^10⁵ 1/M·s or _faster , at about 9.5 × ^10⁵ 1/M·s or _faster , or at about 1 × ^10⁶ 1/M·s or faster. 1/M·s or faster k- _{associations bind} to human PD-L1 and/or PD-L2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that bind to human PD-L1 or PD-L2 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k-dissociation rate of about 2.1 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.3 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, to human PD-1 with _{a k-dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, and to human PD-1 with a k _{-dissociation rate} of about 2.6 × ^{10⁻⁵ 1/s} or slower. It binds to human PD-1 at a k _{dissociation rate} of 1/s or slower, to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 2.7 × ^10⁻⁵ 1/s or slower, or to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, with an IC50 of about 9 nM or less, with an IC50 of about 8 nM or less, with an IC50 of about 7 nM or less, and with an IC50 of about 6 nM or less. It can either inhibit the binding of human PD-L1 or human PD-L2 to human PD-1, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower, or block human PD-1 or block the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-L1 inhibitor is durvalumab, also known as MEDI4736 (available from Medimmune, LLC (a subsidiary of AstraZeneca plc) in Gaithersburg, Maryland), or its antigen-binding fragment, conjugate, or variant. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013/0034559, the disclosure of which is incorporated herein by reference. The clinical efficacy of durvalumab has been described in Page et al., Ann. Rev. Med., 2014, 65, 185-202; Brahmer et al., J. Clin. Oncol. 2014, 32, 5s (Supplement, Abstract 8021); and McDermott et al., Cancer Treatment Rev., 2014, 40, 1056-64. The preparation and characterization of durvalumab are described in U.S. Patent No. 8,779,108, the disclosure of which is incorporated herein by reference. The amino acid sequence of durvalumab is set forth in Table 20. The durvalumab monoclonal antibody comprises disulfide bonds at 22-96, 22”-96”, 23”-89”’, 23”’-89”’, 135”-195”’, 135”’-195”’, 148”-204”, 148”-204”, 215”-224”, 215”’-224”, 230-230”, 233-233”, 265-325”, 265”-325”, 371-429” and 371”-429”’; and N-glycosylation sites at Asn-301” and Asn-301”.

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:178 and the light chain provided in SEQ ID NO:179. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of the durvalumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:180, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:181 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises VH and _{VL regions} , each at least 99% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:182, SEQ ID NO:183, and SEQ ID NO:184, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:185, SEQ ID NO:186, and SEQ ID NO:187, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of anti-PD-L1 approved by a regulatory agency, referencing durvalumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab.

Table 20. Amino acid sequences of PD-L1 inhibitors associated with durvalumab.

In some embodiments, the PD-L1 inhibitor is avelumab, also known as MSB0010718C (available from MerckKGaA/EMD Serono) or its antigen-binding fragment, conjugate, or variant. The preparation and characterization of avelumab are described in U.S. Patent Application Publication No. US2014/0341917 A1, the disclosure of which is incorporated herein by reference. The amino acid sequence of avelumab is set forth in Table 21. Avirumab contains heavy chain intra-chain disulfide bonds (C23-C104) at 22-96, 147-203, 264-324, 370-428, 22”-96”, 147”-203”, 264”-324”, and 370”-428”; and light chain intra-chain disulfide bonds (C23-C104) at 22’-90”, 138’-197”, 22”’-90”’, and 138”’-197”’; 223- Intra-heavy disulfide bonds between the heavy and light chains at 215' and 223"-215"' (h 5-CL 126); intra-heavy disulfide bonds between the heavy and light chains at 229"-229" and 232"-232" (h11, h 14); N-glycosylation sites at 300" and 300" (H CH2 N84.4); fucoidan-like CHO polysaccharides of the fucoidan complex; and H CHS K2 C-terminal lysine cleavage at 450" and 450".

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:188 and the light chain provided in SEQ ID NO:189. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of avirumab. In some embodiments, the heavy chain variable region (VH) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:190, and the light chain variable region (VL) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:191 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:195, SEQ ID NO:196, and SEQ ID NO:197, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of an anti-PD-L1 antibody approved by a pharmaceutical regulatory agency, referencing avirumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. The anti-PD-L1 antibody may be authorized by a pharmaceutical regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is avelumab.

Table 21. Amino acid sequences of PD-L1 inhibitors associated with avirumab.

In some embodiments, the PD-L1 inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (available from Genentech (a subsidiary of Roche Holding AG) in Basel, Switzerland, under the TECENTRIQ license), or an antigen-binding fragment, conjugate, or variant thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent Application Publications Nos. 2010/0203056A1, 2013/0045200A1, 2013/0045201A1, 2013/0045202A1, or 2014/0065135A1, the disclosure of which is specifically incorporated herein by reference. The preparation and characterization of atezolizumab are described in U.S. Patent No. 8,217,149, the disclosure of which is incorporated herein by reference. The amino acid sequence of atezolizumab is set forth in Table 22. Atezolizumab has intrachain disulfide bonds (C23-C104) at 22-96, 145-201, 262-322, 368-426, 22”-96”, 145”-201”, 262”-322”, and 368”-426”; and at 23”-88”, 134”-194”, 23”’-88”’, and 134”’-194”’. Intra-light chain disulfide bonds (C23-C104); intra-heavy chain disulfide bonds at 221-214' and 221”-214”' (h 5-CL 126); intra-heavy chain disulfide bonds at 227-227” and 230-230” (h 11, h 14); and N-glycosylation sites at 298 and 298' (H CH2 N84.4>A).

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:198 and the light chain provided in SEQ ID NO:199. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of atezolizumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:200, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:201 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, _respectively . In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:204, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:205, SEQ ID NO:206, and SEQ ID NO:207, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the anti-PD-L1 antibody is a biosimilar monoclonal antibody of atezolizumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab.

Table 22. Amino acid sequences of PD-L1 inhibitors associated with atezolizumab.

In some embodiments, the PD-L1 inhibitor includes those antibodies described in U.S. Patent Application Publication No. 2014/0341917 A1, the disclosure of which is incorporated herein by reference. In other embodiments, an antibody competing with any of these antibodies for binding to PD-L1 is also included. In some embodiments, the anti-PD-L1 antibody is MDX-1105, also known as BMS-935559, disclosed in U.S. Patent No. 7,943,743, the disclosure of which is incorporated herein by reference. In some embodiments, the anti-PD-L1 antibody is selected from the anti-PD-L1 antibody disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference.

In some embodiments, the PD-L1 inhibitor is a commercially available monoclonal antibody, such as INVIVOMAB anti-m-PD-L1 clone 10F.9G2 (catalog number BE0101, Bio X Cell, West Lebanon, New Hampshire, USA). In some embodiments, the anti-PD-L1 antibody is a commercially available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). Many commercially available anti-PD-L1 antibodies are known to those skilled in the art.

In some implementations, the PD-L2 inhibitor is a commercially available monoclonal antibody, such as BIOLEGEND24F.10C12 mouse IgG2a, κ isotype (catalog number 329602, Biolegend, San Diego, California), SIGMA anti-PD-L2 antibody (catalog number SAB3500395, Sigma-Aldrich, St. Louis, Missouri), or other commercially available anti-PD-L2 antibodies known to those skilled in the art.

2. Combination with CTLA-4 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, including TIL and one or more CTLA-4 inhibitors.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily and is expressed on the surface of helper T cells. CTLA-4 is a negative regulator of CD28-dependent T cell activation and acts as a checkpoint for adaptive immune responses. Similar to the T cell costimulatory protein CD28, CTLA-4 binds to antigens and presents CD80 and CD86 on cells. CTLA-4 delivers inhibitory signals to T cells, while CD28 delivers stimulatory signals. Human antibodies against human CTLA-4 have been described as immunomodulators for many disease conditions, such as for the treatment or prevention of viral and bacterial infections and for the treatment of cancer (WO 01/14424 and WO 00/37504). Several fully human anti-human CTLA-4 monoclonal antibodies (mAbs) have been investigated in clinical trials for the treatment of various types of solid tumors, including (but not limited to) ipilimumab (MDX-010) and trimemumab (CP-675,206).

In some embodiments, the CTLA-4 inhibitor may be any CTLA-4 inhibitor or CTLA-4 blocker known in the art. In particular, it is one of the CTLA-4 inhibitors or blockers described in more detail below. Regarding CTLA-4 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or bioanalyte. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor may also refer to a small molecule compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

CTLA-4 inhibitors suitable for the methods of the present invention include (but are not limited to) anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (ipilimumab), trimemumab, anti-CD28 antibodies, anti-CTLA-4 adenettin, anti-CTLA-4 domain antibodies, and single-chain anti-CTLs. The following are listed: A-4 fragment, heavy chain anti-CTLA-4 fragment, light chain anti-CTLA-4 fragment, CTLA-4 inhibitor activating the co-stimulatory pathway, antibody disclosed in PCT Publication No. WO 2001/014424, antibody disclosed in PCT Publication No. WO 2004/035607, antibody disclosed in U.S. Publication No. 2005/0201994, and antibody disclosed in European Patent No. EP 1212422 B1, the disclosures of each of which are incorporated herein by reference. Other CTLA-4 antibodies are described in U.S. Patents 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publications WO 01/14424 and WO 00/37504; and U.S. Publications 2002/0039581 and 2002/086014, the contents of each of which are incorporated herein by reference. Other anti-CTLA-4 antibodies that may be used in the methods of this invention include, for example, antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (Antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998); and U.S. Patents 5,977,318, 6,682,736, 7,109,003 and 7,132,281, the disclosure of each of which is incorporated herein by reference.

Other CTLA-4 inhibitors include (but are not limited to) the following: those that, by means of activation, disrupt the ability of CD28 antigen to bind to its homologous ligand, inhibit the ability of CTLA-4 to bind to its homologous ligand, enhance T cell responses via the co-stimulatory pathway, disrupt the ability of B7 to bind to CD28 and/or CTLA-4, disrupt the ability of B7 to activate the co-stimulatory pathway, disrupt the ability of CD80 to bind to CD28 and/or CTLA-4, disrupt the ability of CD80 to activate the co-stimulatory pathway, disrupt the ability of CD86 to bind to CD28 and/or CTLA-4, disrupt the ability of CD86 to activate the co-stimulatory pathway, and any inhibitor that disrupts the co-stimulatory pathway. This must include: small molecule inhibitors of CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antibodies against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antisense molecules against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; adenettin against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; RNAi inhibitors (single-stranded and double-stranded) against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; and other CTLA-4 inhibitors.

In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 at a K d , which is about 10  -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, for example, between 10 -13 M and 10 -16 M, or within any range with any two of the foregoing values as endpoints. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is no more than 10 times that of the Kd of ipilimumab. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is substantially the same as or less than that of ipilimumab (e.g., up to 10 times or up to 100 times lower). In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is no more than 10-fold higher than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively. In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is approximately the same as or less than (e.g., up to 10-fold or up to 100-fold lower) than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively.

In some embodiments, a CTLA-4 inhibitor is used in an amount sufficient to inhibit CTLA-4 expression and/or reduce the biological activity of CTLA-4 by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100%. In some embodiments, a CTLA-4 pathway inhibitor is used in an amount sufficient to reduce the biological activity of CTLA-4 by reducing the binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100% relative to a suitable control. In the context of assessing or quantifying the effect of an agent of interest, a suitable control is typically a biological system (e.g., cells or a subject) that has not been exposed to the agent of interest (e.g., a CTLA-4 pathway inhibitor) or has been treated with the agent (or has been exposed to or treated with a negligible amount). In some embodiments, the biological system may serve as its own control, for example, by assessing the biological system before exposure to or treatment with the agent and comparing its status after the start or end of exposure or treatment. In some embodiments, a historical control may be used.

In some embodiments, the CTLA-4 inhibitor is ipilimumab (available from Bristol-Myers Squibb at Yervoy) or its biosimilars, antigen-binding fragments, conjugates, or variants. As known in the art, ipilimumab refers to an anti-CTLA-4 antibody, a fully human IgG 1κ antibody derived from transgenic mice having human genes encoding both heavy and light chains to produce a functional human lineage. Ipilimumab is also mentioned in its CAS Registry No. 477202-00-9 and in PCT Publication No. WO 01/14424, which is incorporated herein by reference in its entirety for all purposes. It is disclosed in the form of Antibody 10DI. Specifically, ipilimumab contains a light chain variable region and a heavy chain variable region (having a light chain variable region comprising SEQ ID NO: 211 and a heavy chain variable region comprising SEQ ID NO: 210). Pharmaceutical compositions of ipilimumab comprise all pharmaceutically acceptable compositions containing ipilimumab and one or more diluents, mediators, or excipients. Examples of pharmaceutical compositions containing ipilimumab are described in International Patent Application Publication No. WO 2007/67959. Ipilimumab can be administered intravenously (IV).

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:208 and the light chain provided in SEQ ID NO:209. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of ipilimumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:210, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:211 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:215, SEQ ID NO:216, and SEQ ID NO:217, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to ipilimumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of ipilimumab is illustrated in Table 23. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab.

Table 23. Amino acid sequence of ipilimumab.

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, ipilimumab administration may be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, ipilimumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab is administered at approximately mg/kg every 3 weeks for up to 4 consecutive doses to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered as adjuvant therapy for melanoma. In some implementations, ipilimumab is administered at approximately 10 mg/kg every 3 weeks for four doses, followed by 10 mg/kg every 12 weeks for up to 3 years as adjuvant therapy for melanoma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat advanced renal cell carcinoma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 3 weeks, followed by nivolumab at 3 mg/kg on the same day, for a total of 4 doses to treat advanced renal cell carcinoma. In some implementations, after completing the combined 4-dose regimen, nivolumab can be administered as a single agent against advanced renal cell carcinoma and/or renal cell carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, ipilimumab is administered intravenously at approximately 1 mg/kg every 30 minutes over 3 weeks, followed immediately by nivolumab intravenously at 3 mg/kg every 30 minutes on the same day, for a total of 4 doses to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, after completing 4 doses of the composition, nivolumab is administered as a single agent for microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, as recommended according to standard dosing regimens. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also begin 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat hepatocellular carcinoma. In some implementations, ipilimumab is administered intravenously at approximately 3 mg/kg every 3 weeks over 30 minutes, followed immediately by nivolumab intravenously at 1 mg/kg over 30 minutes on the same day, for a total of four doses to treat hepatocellular carcinoma. In some implementations, after completing the combined four-dose regimen, nivolumab is administered as a single agent against hepatocellular carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, followed by nivolumab at 3 mg/kg every 2 weeks to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, plus 360 mg nivolumab every 3 weeks for 2 cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat malignant pleural mesothelioma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks and nivolumab at 360 mg every 3 weeks to treat malignant pleural mesothelioma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

Trimelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has CAS number 745013-59-6. Trimelimumab is disclosed in U.S. Patent No. 6,682,736 (incorporated herein by reference) as antibody 11.2.1. The amino acid sequences of the heavy and light chains of trimemumab are described in SEQ IND NO: 218 and 219, respectively. Trimelimumab has been studied in clinical trials for the treatment of various tumors, including melanoma and breast cancer; wherein it is administered intravenously in single or multiple doses at a dose range between 0.01 and 15 mg/kg every 4 or 12 weeks. In the embodiments provided by the present invention, trimemumab is administered topically, particularly intradermally or subcutaneously. The effective amount of trimemumab administered intradermally or subcutaneously is typically in the range of 5-200 mg/dose per person. In some implementations, the effective dose of trimemumab is in the range of 10-150 mg/dose per person. In some specific implementations, the effective dose of trimemumab is approximately 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175 or 200 mg/dose per person.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:218 and the light chain provided in SEQ ID NO:219. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of trimexumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:220, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:221 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in _SEQ ID NO:220 and _SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:225, SEQ ID NO:226, and SEQ ID NO:227, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a biosimilar monoclonal antibody of anti-CTLA-4 approved by a regulatory agency, referencing trimemumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of trimemumab is illustrated in Table 24. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab.

Table 24. Amino acid sequence of trimemumab.

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, wherein trimemumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, trimemumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, trimemumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is zeflimab from Agenus or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Zeflimab is a fully human monoclonal antibody. Zeflimab is designated Chemical Abstracts Service (CAS) Registry No. 2148321-69-9 and is also known as AGEN1884. The preparation and characterization of zeflimab are described in U.S. Patent No. 10,144,779 and U.S. Patent Application Publication No. US2020/0024350A1, the disclosures of which are incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:228 and the light chain provided in SEQ ID NO:229. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of zeflimab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:230, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:231 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:231, SEQ ID NO:233, and SEQ ID NO:234, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:235, SEQ ID NO:236, and SEQ ID NO:237, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to zeflimab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of zeflimab is illustrated in Table 25. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab.

Table 25. Amino acid sequence of zefrizinumab.

Other examples of anti-CTLA-4 antibodies include (but are not limited to): AAGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659, and ADG116, which are known to those skilled in the art.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent disclosures: US2019/0048096 A1; US2020/0223907; US2019/0201334; US2019/0201334; US2005/0201994; EP 1212422B1; WO 2018/204760; WO 2018/204760; WO 2001/014424; WO2 WO 2004/035607; WO 2003/086459; WO 2012/120125; WO 2000/037504; WO 2009/100140; WO2006/09649; WO2005092380; WO 2007/123737; WO 2006/029219; WO 2010/0979597; WO2006/12168; and WO1997020574, each of which is incorporated herein by reference. Other CTLA-4 antibodies are described in the following: U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227 and 6,984,720; PCT Publications Nos. WO 01/14424 and WO 00/37504; and U.S. Publications Nos. 2002/0039581 and 2002/086014; and/or U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281, each of which is incorporated herein by reference. In some embodiments, the anti-CTLA-4 antibody is, for example, one of the antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 10067-10071 (1998); Camacho et al., J. Clin. Oncol., 2004, 22, 145 (Abstract No. 2505 (2004) (Antibody CP-675206); or Mokyr et al., Cancer Res., 1998, 58, 5301-5304 (1998), each of which is incorporated herein by reference.

In some implementations, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996/040915 (incorporated herein by reference).

In some implementations, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, anti-CTLA-4 RNAi molecules may be in the form described in: PCT Publications WO 1999/032619 and WO 2001/029058; U.S. Publications 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, and 2005/01009. Patent Nos. 13, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443; and/or U.S. Patent Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in European Patent No. 1309726 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in U.S. Patent Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In some embodiments, the CTLA-4 inhibitor is an aptamer described in International Patent Application Publication No. WO2004/081021 (incorporated herein by reference).

In other embodiments, the anti-CTLA-4 RNAi molecule of the present invention is the RNA molecule described in U.S. Patent Nos. 5,898,031, 6,107,094, 7,432,249 and 7,432,250 and European Application No. EP 0928290 (incorporated herein by reference).

3. Preconditioning of lymphocyte depletion in patients

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the present disclosure. In some embodiments, the present invention includes a TIL population for treating cancer in patients pretreated with non-myeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/ ^m² /day for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion (day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 (aldeleukin, available from PROLEUKIN) every 8 hours at 720,000 IU/kg to achieve physiological tolerance. In some implementations, TIL groups are used in combination with IL-2 to treat cancer, wherein IL-2 is administered after the TIL group.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system ('cytokine pool') prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as "immunosuppressive modulation") in a patient prior to the introduction of the TIL of the present invention.

Generally, lymphocyte depletion is achieved by administration of fludarabine or cyclophosphamide (the active form of which is called equiphosphamide) and combinations thereof. Such methods are described in Gassner et al., Cancer Immunol. Immunother. 2011, 60, 75-85; Muranski et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-5239; and Dudley et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL. In some embodiments, fludarabine is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, fludarabine is administered at doses of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2–7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4–5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4–5 days.

In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 1 μg/mL. In some embodiments, cyclophosphamide is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, cyclophosphamide is administered at doses of 100 mg/m²/day, 150 mg/m²/day, 175 mg/m²/day, 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day, 275 mg/m²/day, or 300 mg/m²/day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.e., i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2–7 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4-5 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4 days.

In some implementations, lymphocyte depletion is achieved by administering fludarabine and cyclophosphamide together to the patient. In some implementations, fludarabine is administered intravenously at 25 mg/m²/day for 4 days, and cyclophosphamide is administered intravenously at 250 mg/m²/day.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days and fludarabine at a dose of 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered for the first two days and lymphocyte depletion is achieved over a total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 15 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by fludarabine at a dose of 25 mg/m²/day for three days.

In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, mesna is administered at a dose of 15 mg/kg. In some embodiments, mesna is infused, and if continuous infusion is initiated over 24 hours, with the respective cyclophosphamide dose starting, mesna may be infused with cyclophosphamide over approximately 2 hours (days -5 and/or -4), and then infused at a rate of 3 mg/kg/hour for the remaining 22 hours.

In some implementations, lymphocyte depletion includes the following steps: starting the day after administering the third TIL group to the patient, treating the patient with an IL-2 regimen.

In some implementations, lymphocyte depletion includes the following steps: starting on the day the third TIL group is administered to the patient, the patient is treated with an IL-2 regimen.

In some embodiments, lymphocyte depletion comprises a 5-day preconditioning treatment. In some embodiments, the days are indicated as day-5 to day-1, or day 0 to day 4. In some embodiments, the regimen comprises cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises 60 mg/kg intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, the regimen also comprises fludarabine. In some embodiments, the regimen also comprises intravenous fludarabine. In some embodiments, the regimen also comprises 25 mg/ ^m² intravenous fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine at a dose of 25 mg/ ^m² on days -5 and ^-1 (i.e., days 0 to 4).

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for one day.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 26.

Table 26. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 27.

Table 27. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 28.

Table 28. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 29.

Table 29. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day			X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 30.

Table 30. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 31.

Table 31. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 32.

Table 32. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 33.

Table 33. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day			X	X	X
TIL infusion						X

In some embodiments, the TIL infusion used in conjunction with the above-described myeloablative lymphocyte depletion regimens may be any TIL composition described herein, as well as an IL-2 addition regimen and administration of combination therapies as described herein (e.g., PD-1 and PD-L1 inhibitors).

4. IL-2 scheme

In some implementations, the IL-2 regimen includes a high-dose IL-2 regimen comprising alephrine or a biosimilar or variant thereof, administered intravenously starting the day after the effective fraction of treatment in the therapeutic TIL population. Alephrine or a biosimilar or variant thereof is administered as a 15-minute bolus intravenous infusion every eight hours at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient weight) until tolerated, for a maximum of 14 doses. After a 9-day rest period, this course can be repeated for another 14 doses, for a maximum total of 28 doses. In some implementations, IL-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In some implementations, IL-2 is administered at a maximum dose of up to 6 doses.

In some implementations, the IL-2 regimen comprises a tapering IL-2 regimen. Tapering IL-2 regimens have been described in O'Day et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In some implementations, tapering IL-2 therapy comprises an intravenous administration of 18 × ^10⁶ IU/ ^m² over 6 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 12 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 24 hours, followed by an intravenous administration of 4.5 × ^10⁶ IU/ ^m² of adeleukin or a biosimilar or variant thereof over 72 hours. This treatment cycle may be repeated every 28 days for a maximum of four cycles. In some implementations, the decreasing IL-2 regimen includes 18,000,000 IU/ ^m² on day 1, 9,000,000 IU/ ^m² on day 2, and 4,500,000 IU/ ^m² on days 3 and 4.

In some implementations, the IL-2 regimen includes a low-dose IL-2 regimen. Any low-dose IL-2 regimen known in the art may be used, including those described in Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann et al., Lancet Diabetes Endocrinol. 2013, 1, 295-305; and Rosenzwaig et al., Ann. Rheum. Dis. 2019, 78, 209-217, the disclosures of which are incorporated herein by reference. In some implementations, a low-dose IL-2 regimen comprises 18 × ^10⁶ IU/ ^m² of interleukin or its biosimilar or variant every 24 hours, administered as a continuous infusion for 5 days; followed by a 2–6 day period without IL-2 therapy; optionally followed by intravenous administration of interleukin or its biosimilar or variant at 18 × ^10⁶ IU/ ^m² every 24 hours for another 5 days; optionally followed by a 3-week period without IL-2 therapy, after which other cycles may be administered.

In some implementations, IL-2 is administered at a maximum dose of up to 6 doses. In some implementations, high-dose IL-2 regimens are modified for pediatric use. In some implementations, interleukin is administered at a dose of 600,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 400,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 300,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 200,000 IU/kg every 8-12 hours, for a maximum of 6 doses. In some implementations, interleukin is administered at a dose of 100,000 IU/kg every 8-12 hours, for a maximum of 6 doses.

In some embodiments, the IL-2 regimen comprises administering PEGylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bepedipine interleukin or a fragment, variant, or bioanalyte thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen comprises administering THOR-707 or a fragment, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen includes administration of Nevorin Alpha or a fragment, variant, or biosimilar thereof following TIL administration. In some implementations, Nevorin Alpha is administered to the patient at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 protocol includes administering an IL-2 fragment grafted onto the antibody backbone. In some embodiments, the IL-2 protocol includes administering an antibody cytokine graft protein that binds to a low-affinity IL-2 receptor. In some embodiments, the antibody cytokine graft protein includes a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof grafted into the CDR of _VH or _VL , wherein the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof, which is transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody or a fragment thereof, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days, wherein the antibody comprises a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

In some implementations, the serum half-life of the antibody cytokine transplanted protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to interleukin or comparable molecules).

In some embodiments, the TIL infusion used in conjunction with the aforementioned embodiments of the myeloablative lymphocyte depletion regimen may be any TIL composition described herein and may also include MIL and PBL infusions in lieu of TIL infusion, as well as the addition of an IL-2 regimen and administration of combination therapies as described herein (e.g., PD-1 and/or PD-L1 inhibitors and/or CTLA-4 inhibitors).

Implementation Plan

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating the fourth TIL population takes about 9 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 bead agonist or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about one day.

In some implementations, the process of cultivating the fourth TIL population takes about 2 days.

In some implementations, the process of cultivating a fourth TIL population takes about 3 days.

In some implementations, the process of cultivating the fourth TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing a third TIL population takes about 8 days.

In some implementations, the process of culturing a third TIL population takes approximately 9 days.

In some implementations, the process of culturing the third TIL population takes about 8-9 days.

In some implementations, the process of cultivating a third TIL population takes about 10 days.

In some implementations, the process of culturing a third TIL population takes approximately 8-10 days.

In some implementations, the process of culturing a third TIL population takes approximately 11 days.

In some implementations, the process of culturing a third TIL population takes approximately 12 days.

In some implementations, the process of cultivating a third TIL population takes approximately 13 days.

In some implementations, the process of culturing a third TIL population takes approximately 14 days.

In some implementations, the process of cultivating a third TIL population takes about 15 days.

In some implementations, the step of culturing a third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about one day.

In some implementations, the process of cultivating a third TIL population takes about 2 days.

In some implementations, the process of cultivating a third TIL population takes about 3 days.

In some implementations, the process of cultivating a third TIL population takes about 4 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) Culture the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) A fourth population of TILs was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately nine days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a third cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a fourth culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the step of culturing a third or fourth TIL population takes about one day.

In some implementations, the process of culturing a third or fourth TIL population takes about 2 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 3 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 4 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 5 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 6 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, during the step of culturing a first TIL population in a first culture medium, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in the first culture medium.

In some embodiments, during the step of culturing a second TIL population in a second culture medium, the second culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in a second culture medium.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of a first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the initiation of the first amplification takes approximately one day.

In some implementations, the initiation of the first amplification takes approximately 2 days.

In some implementations, the initiation of the first amplification takes approximately 3 days.

In some implementations, the initiation of the first amplification takes approximately 4 days.

In some implementations, the initiation of the first amplification takes approximately 5 days.

In some implementations, the initiation of the first amplification takes approximately 6 days.

In some implementations, the initiation of the first amplification takes approximately 7 days.

In some implementations, the initiation of the first amplification takes approximately 8 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the rapid second amplification takes approximately 9 days.

In some implementations, the rapid second amplification takes approximately 10 days.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, all steps are completed within a period of approximately 16 days.

In some implementations, all steps are completed within a period of approximately 17 days.

In some implementations, all steps are completed within a period of approximately 18 days.

In some implementations, all steps are completed within a period of approximately 18-22 days.

In some implementations, all steps are completed within a period of approximately 19 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 21 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, the first culture medium contains APC.

In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

In some embodiments, the first culture medium contains OKT-3.

In some implementations, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies during the initial amplification step.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digesting tumor tissue samples in an enzyme medium to produce tumor digests;

(c) The tumor digest is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (f), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the first amplification takes about 3 days.

In some implementations, the first amplification takes about 4 days.

In some implementations, the first amplification takes about 5 days.

In some implementations, the first amplification takes about 6 days.

In some implementations, the first amplification takes about 7 days.

In some implementations, the first amplification takes about 8 days.

In some implementations, the first amplification takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes about 5 days.

In some implementations, the second amplification takes about 6 days.

In some implementations, the second amplification takes about 7 days.

In some implementations, the second amplification takes approximately 8 days.

In some implementations, the second amplification takes approximately 9 days.

In some implementations, the second amplification takes about 8-9 days.

In some implementations, the second amplification takes about 10 days.

In some implementations, the second amplification takes about 8-10 days.

In some implementations, the second amplification takes approximately 11 days.

In some implementations, the second amplification takes approximately 12 days.

In some implementations, the second amplification takes approximately 13 days.

In some implementations, the second amplification takes approximately 14 days.

In some implementations, the second amplification takes about 15 days.

In some implementations, the second amplification is performed through the following steps:

(i) The first period of culturing the fourth TIL population in the second culture medium for about 5 days.

(ii) The culture from step (i) is subdivided into multiple subcultures, each of which is transferred to a separate sealed container providing a third breathable surface and cultured for a second period of approximately 4 or 5 days in a third medium containing IL-2, wherein the transition from step (i) to step (ii) is performed without opening the system, and

(iii) Multiple subcultures are merged to produce a fifth TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, all steps are completed within a period of approximately 25 days.

In some implementations, all steps are completed within a period of approximately 26 days.

In some implementations, all steps are completed within a period of approximately 27 days.

In some implementations, all steps are completed within a period of approximately 28 days.

In some implementations, all steps are completed within a period of approximately 29 days.

In some implementations, all steps are completed within a period of approximately 30 days.

In some implementations, all steps are completed within a period of approximately 31 days.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Digesting a first TIL population from tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a patient or subject in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, this document provides expanded numbers of tumor-infiltrating lymphocytes (TILs) or therapeutic TIL populations produced by the methods described herein.

In some embodiments, this document provides pharmaceutical compositions comprising an amplified number of TILs or a therapeutic population of TILs as described herein and a pharmaceutically acceptable carrier.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of an expanded number of TILs or a therapeutically active population of TILs as described herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor excised from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor digest to a closed system and performing a first amplification to produce a second TIL population by culturing the first TIL population in a first cell culture medium containing IL-2, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting PD-1 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CISH expression.

In some implementations, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting LAG-3 expression and a TALEN system for inhibiting TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is approximately 2.3 × 10¹⁰ to approximately 13.7 × 10¹⁰ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of TIL cells in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the administration of TIL cells to the patient in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Example

The embodiments covered herein are now described with reference to the following examples. These examples are provided for illustrative purposes only and this disclosure should in no way be construed as limiting to these examples, but rather as covering any and all variations that become apparent from the teachings provided herein.

Example 1: Preparation of culture medium for pre-REP and REP processes

This embodiment describes a procedure for preparing a tissue culture medium suitable for cultures involving tumor-infiltrating lymphocytes (TILs) derived from various solid tumors. This culture medium can be used to prepare any of the TILs described in this application and other embodiments.

Prepare CM1 . Remove the following reagents from the freezer and warm them in a 37°C water bath: (RPMI 1640, human AB serum, 200 mM L-glutamine). Prepare CM1 medium by adding each component to the top of a 0.2 μm filter unit suitable for the desired filtration volume, according to Table 34 below. Store at 4°C.

Table 34. Preparation of CM1

On the day of use, preheat the required amount of CM1 in a 37°C water bath and add 6000 IU/mL IL-2.

Additional information may be added as needed, based on Table 35.

Table 35. Additional information for CM1 as needed.

Preparation of CM2

Remove the prepared CM1 from the refrigerator or prepare fresh CM1. Remove AIM- from the refrigerator and prepare the required amount of CM2 by mixing the prepared CM1 with an equal volume of AIM- in a sterile culture medium bottle. On the day of use, add 3000 IU/mL IL-2 to the CM2 culture medium. On the day of use, prepare a sufficient amount of CM2 with 3000 IU/mL IL-2. Label the CM2 culture medium bottle with the name, the initials of the preparer's name, the filtration/preparation date, and the two-week expiry date, and store at 4°C until needed for tissue culture.

Preparation of CM3

Prepare CM3 on the day it is needed. CM3 is the same as AIM-medium, but supplemented with 3000 IU/mL IL-2 on the day of use. Prepare the amount of CM3 needed for the experiment by directly adding the IL-2 stock solution to the AIM-V bottle or bag. Mix thoroughly by gentle shaking. Immediately after adding to AIM-V, label the bottle “3000 IU/mL IL-2”. If there is excess CM3, store it in a bottle at 4°C, labeling it with the medium name, the preparer's initials, the date of preparation, and its expiration date (7 days after preparation). After storing at 4°C for 7 days, discard the medium supplemented with IL-2.

Preparation of CM4

CM4 is the same as CM3, but with an additional 2 mM GlutaMAX ^™ (final concentration). Add 10 mL of 200 mM GlutaMAX ^™ per 1 L of CM3. Prepare the required amount of CM4 by directly adding IL-2 and GlutaMAX ^™ stock solution to the AIM-V bottle or bag. Mix thoroughly with gentle shaking. Immediately after adding to the AIM-V, label the bottle “3000 IL/mL IL-2 and GlutaMAX”. If excess CM4 exists, store it in the bottle at 4°C, labeling it with the culture medium name, “GlutaMAX”, and its expiration date (7 days after preparation). Discard the culture medium supplemented with IL-2 after storage at 4°C for more than 7 days.

Example 2: Use of a mixture of IL-2, IL-15 and IL-21 cytokines

This embodiment describes the use of IL-2, IL-15, and IL-21 cytokines, which act as additional T cell growth factors, in combination with the TIL process of any embodiment herein.

Using the procedures described herein, at the start of culture, TILs were observed to grow from tumors in one experimental group in the presence of IL-2 and in another group in the presence of a combination of IL-2, IL-15, and IL-21, which substituted for IL-2. At the completion of pre-REP, the amplification, phenotype, function (CD107a+ and IFN-γ), and TCR Vβ lineage of the cultures were assessed. IL-15 and IL-21 are described elsewhere in this paper and in Santegoets et al., J. Transl. Med., 2013, 11, 37.

The results showed enhanced TIL amplification (>20%) in CD4 ⁺ and CD8 ⁺ cells under IL-2, IL-15, and IL-21 treatments compared to IL-2-only conditions. Significant skewness towards a CD8 ⁺ population with a skewed TCR Vβ lineage was observed in TILs obtained from IL-2, IL-15, and IL-21-treated cultures compared to IL-2-only cultures. IFN-γ and CD107a levels were increased in TILs treated with IL-2, IL-15, and IL-21 compared to IL-2-only TILs.

Example 3: Quantification of individual batches of γ-irradiated peripheral mononuclear cells

This embodiment describes a simplified procedure for quantifying individual batches of γ-irradiated peripheral monocytes (PBMCs, also known as monocytes or MNCs) used as allogeneic feeder cells in the exemplary methods described herein.

Each batch of irradiated MNC feeder cells was prepared from individual donors. Each batch or donor was individually screened based on its ability to amplify TILs in the REP in the presence of purified anti-CD3 (clonal OKT3) antibody and interleukin-2 (IL-2). Furthermore, each batch of feeder cells was tested without the addition of TILs to verify that the received gamma irradiation dose was sufficient to prevent replication.

TIL regeneration requires γ-irradiated, growth-arrested MNC feeder cells. Membrane receptors on the feeder MNCs bind to anti-CD3 (clonal OKT3) antibodies and cross-link with TILs in the REP culture flask, stimulating TIL expansion. Feeder cell batches are prepared by leukocyte removal from whole blood obtained from individual donors. The leukocyte removal product is centrifuged, washed, irradiated, and cryopreserved under GMP conditions on a Ficoll-Hypaque apparatus.

Importantly, live feeder cells should not be infused into patients receiving TIL therapy, as this can cause graft-versus-host disease (GVHD). Consequently, feeder cell growth is inhibited by gamma irradiation, leading to double-strand DNA breaks and loss of MNC cell viability upon reculture.

Feeder cell batches were evaluated according to two criteria: (1) their ability to amplify TILs by >100-fold in co-culture, and (2) their insufficient replication capacity.

Feeder cell batches were tested in a mini-REP format using two primary pre-REP TIL lines grown in vertical T25 tissue culture flasks. Feeder cell batches were tested against two distinct TIL lines, each with a unique ability to proliferate in response to activation within the REP. As a control, a number of previously demonstrated irradiated MNC feeder cells meeting the above criteria were processed with the test batches.

A stock solution of the same pre-REP TIL line is available to test all conditions and all feeder cell batches, ensuring that all batches tested in a single experiment undergo equivalent testing.

For each batch of feeder cells tested, a total of six T25 culture flasks were used: pre-REP TIL line #1 (2 flasks); pre-REP TIL line #2 (2 flasks); and feeder cell control (2 flasks). Flasks containing TIL lines #1 and #2 were used to evaluate the feeder cell batch's ability to amplify TILs. The feeder cell control flasks were used to evaluate insufficient replication capacity in the feeder cell batch.

A. Experimental Protocol

On day -2/3, thaw the TIL culture . Prepare CM2 medium and warm it in a 37°C water bath. Prepare 40 mL of CM2 supplemented with 3000 IU/mL IL-2. Keep warm until used. Place 20 mL of pre-warmed CM2 (without IL-2) into each of two 50 mL conical tubes labeled with the name of the TIL culture used. Remove the two designated pre-REP TIL cultures from the LN2 reservoir and transfer the vials to the tissue culture room. Thaw the vials by placing them in a sealed zip-lock storage bag in a 37°C water bath until a small amount of ice remains.

Using a sterile pipette, immediately transfer the contents of each vial to 20 mL of CM2 in a prepared, labeled 50 mL conical tube. Make up to 40 mL with IL-2-free CM2 to wash the cells and centrifuge at 400×CF for 5 minutes. Aspirate the supernatant and resuspend the cells in 5 mL of warm CM2 supplemented with 3000 IU/mL IL-2.

Take two aliquots (20 μL each) for cell counting using an automated cell counter. Record the counts. During counting, place a 50 mL conical tube containing TIL cells in a humidified 37°C, 5% _CO2 incubator with the cap loosened to allow gas exchange. Determine the cell concentration and dilute the TIL to 1 × ^10⁶ cells/mL in CM₂ supplemented with 3000 IU/mL IL-2.

In a humidified 37°C incubator, culture at 2 ml/well in multiple wells of a 24-well tissue culture plate as needed until day 0 of the micro-REP. Culture different TIL lines in separate 24-well tissue culture plates to avoid confusion and potential cross-contamination.

Day 0, start the micro-REP . Prepare sufficient CM2 medium for the number of feeder cell batches to be tested. (For example, prepare 800 mL of CM2 medium for testing 4 feeder cell batches at once). Divide a portion of the CM2 prepared above into equal parts and supplement it with 3000 IU/mL IL-2 for cell culture. (For example, prepare 500 mL of CM2 medium with 3000 IU/mL IL-2 for testing 4 feeder cell batches at once).

To prevent cross-contamination, the 24-well plates containing TIL cultures were removed from the incubator and transferred to the BSC.

Using sterile pipettes or 100-1000 μL pipettes and tips, remove approximately 1 mL of culture medium from each well to be used and place it into an unused well of a 24-well tissue culture plate.

Using fresh, sterile pipettes or 100-1000 μL pipettes and tips, mix the remaining culture medium with the TIL in the wells to resuspend the cells, and then transfer the cell suspension to 50 mL conical tubes labeled with the TIL batch name and record the volume.

Wash each well with the reserved culture medium and transfer the volume to the same 50 mL conical tube. Rotate the cells at 400 × CF to collect the cell pellet. Drain the culture supernatant and resuspend the cell pellet in 2–5 mL of CM2 medium containing 3000 IU/mL IL-2. The volume to be used is based on the number of wells collected and the size of the pellet; that is, the volume should be sufficient to ensure a concentration >1.3 × ^10⁶ cells/mL.

Using a serum pipette, thoroughly mix the cell suspension and record the volume. Take 200 μL for cell counting using an automated cell counter. During counting, place a 50 mL conical tube containing TIL cells in a humidified 5% _CO2 incubator at 37°C with the cap loosened to allow gas exchange. Record the count.

Remove the 50 mL conical tube containing TIL cells from the incubator and resuspend the cells at a concentration of 1.3 × ^10⁶ cells/mL in warm CM2 supplemented with 3000 IU/mL IL-2. Place the 50 mL conical tube back into the incubator and loosen the cap.

For the second TIL system, repeat the above steps.

Before plating TIL into the T25 culture flasks used for experiments, dilute TIL 1:10 to a final concentration of 1.3 × ^10⁵ cells/mL as shown below.

Prepare a working solution of pure CD3 (OKT3) from MACS GMP . Remove the OKT3 stock solution (1 mg/mL) from the 4°C freezer and place it in BSC. Use OKT3 at a final concentration of 30 ng/mL in the micro-REP medium.

In each T25 culture flask used in the experiment, 600 ng OKT3 is required per 20 mL; this is equivalent to 60 μL of 10 μg/mL solution per 20 mL, or 360 μL for all 6 culture flasks tested for each feeder cell batch.

For each feeder cell batch tested, prepare 400 μL of a 1:100 dilution of 1 mg/mL OKT3 for a working concentration of 10 μg/mL (e.g., for a single test of 4 feeder cell batches, prepare 1600 μL of a 1:100 dilution of 1 mg/mL OKT3: 16 μL of 1 mg/mL OKT3 + 1.584 mL of CM2 medium with 3000 IU/mL IL-2).

Prepare T25 culture flasks . Label each culture flask and fill it with CM2 medium before preparing feeder cells. Place the flasks in a humidified 5% _CO2 incubator at 37°C to keep the medium warm while awaiting the addition of the remaining components. After preparing feeder cells, add the components to the CM2 in each culture flask.

Other information is provided in Table 36.

Table 36. Solution Information.

Prepare feeder cells . For this protocol, each batch tested requires at least 78 × ^10⁶ feeder cells. Each 1 mL vial frozen by SDBB has 100 × ^10⁶ viable cells at freezing. Assuming a 50% recovery rate after thawing from a liquid _N₂ reservoir, it is recommended to thaw at least two 1 mL vials of feeder cells per batch, thus using an estimated 100 × ^10⁶ viable cells per REP. Alternatively, if supplied in 1.8 mL vials, a single vial may provide sufficient feeder cells.

Before thawing the feeder cells, pre-warm approximately 50 mL of IL-2-free CM2 for each feeder cell batch to be tested. Remove the designated feeder cell batch vial from the LN2 reservoir, place it in a zip-lock storage bag, and keep it on ice. Thaw the vial in the sealed zip-lock storage bag by immersion in a 37°C water bath. Remove the vial from the zip-lock bag, spray or wipe it with 70% EtOH, and transfer it to a BSC.

Using a pipette, immediately transfer the contents of the feeder cell vial to 30 mL of warm CM2 in a 50 mL conical tube. Wash the vial with a small volume of CM2 to remove any residual cells and centrifuge at 400×CF for 5 min. Aspirate the supernatant and resuspend the cells in 4 mL of warm CM2 with 3000 IU/mL IL-2. Take 200 μL for cell counting using an automated cell counter. Record the count.

Cells were resuspended at 1.3 × ^10⁷ cells/mL in warm CM2 with 3000 IU/mL IL-2. TIL cells were diluted from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL.

Set up the co-culture . Dilute TIL cells from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL. Add 4.5 mL of CM2 medium to a 15 mL conical tube. Remove the TIL cells from the incubator and resuspend them thoroughly using a 10 mL serum pipette. Take 0.5 mL of the 1.3 × ^10⁶ cells/mL TIL suspension and add it to 4.5 mL of medium in a 15 mL conical tube. Return the TIL stock solution vial to the incubator. Mix thoroughly. Repeat the above steps for the second TIL line.

Transfer the culture flasks containing pre-warmed medium for a single feeder cell batch from the incubator to the BSC. Mix the feeder cells by pipetting several times up and down with a 1 mL pipette tip, and transfer 1 mL (1.3 × ^10⁷ cells) to each culture flask containing the feeder cell batch. Add 60 μL of OKT3 working stock solution (10 μg/mL) to each culture flask. Return the two control flasks to the incubator.

Transfer 1 mL (1.3 × ^10⁵ ) of each TIL batch to a correspondingly labeled T25 culture flask. Place the flask back into the incubator and incubate upright. Begin the intervention on day 5. Repeat this procedure for all feeder cell batches tested.

On day 5, the culture medium was changed . CM2 with 3000 IU/mL IL-2 was prepared. 10 mL was needed for each culture flask. Using a 10 mL pipette, 10 mL of warm CM2 with 3000 IU/mL IL-2 was transferred to each culture flask. The culture flasks were returned to the incubator and incubated upright until day 7. The above procedure was repeated for all tested feeder cell batches.

Day 7, collect the culture . Remove the culture flasks from the incubator and transfer them to BSC, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove 10 mL of culture medium from each test flask and 15 mL of culture medium from each control flask.

Using a 10 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using the appropriate standard operating procedure with an automated cell counter. Record the counts on day 7. Repeat this procedure for all feeder cell batches tested.

The replication capacity of the feeder cell control flasks was insufficient, and the fold expansion of the culture flasks containing TILs was evaluated starting from day 0.

On day 7, continue processing the feeder cell control bottles until day 14. After counting the feeder cells on day 7, add 15 mL of fresh CM2 medium containing 3000 IU/mL IL-2 to each control bottle. Return the control bottles to the incubator and incubate them upright until day 14.

Day 14, extended non-proliferative phase of the feeder cell control flasks . Remove the culture flasks from the incubator and transfer them to BSCs, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove approximately 17 mL of culture medium from each control flask. Using a 5 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. Record the volume of each culture flask.

After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using an automated cell counter device following the appropriate standard operating procedure and record the count. Repeat this procedure for all feeder cell batches tested.

B. Results and Acceptance Criteria

Results . The dose of gamma irradiation was sufficient to prevent feeder cells from replicating. All batches were expected to meet the evaluation criteria and also showed a reduction in the total number of viable feeder cells remaining on day 7 of REP culture compared to day 0. All feeder cell batches were expected to meet the following evaluation criteria: 100-fold growth expansion of TILs up to day 7 of REP culture. The feeder cell control flask counts on day 14 were expected to continue the non-proliferative trend visible on day 7.

Acceptance Criteria . Each replicated TIL line for each batch of feeder cells must meet the following acceptance criteria. The acceptance criteria are doubled, as shown in Table 37 below.

Table 37. Implementation plan for acceptance criteria.

The study evaluated whether the irradiation dose was sufficient to prevent MNC feeder cells from replicating when cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. Insufficient replication capacity was assessed by total live cell count (TVC) as determined by automated cell counting at days 7 and 14 of the repetitive period (REP).

The acceptance criterion was "no growth," meaning that on days 7 and 14, the total number of viable cells did not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP.

The ability of feeder cells to support TIL expansion was evaluated. TIL growth was measured by the fold increase in live cells from day 0 of the Reproductive Phase (REP) to day 7 of the REP. On day 7, the TIL culture reached a minimum 100-fold expansion (i.e., more than 100 times the total number of live TIL cells introduced into the culture on day 0 of the REP), as evaluated by automated cell counting.

Emergency testing of MNC feeder cell batches that do not meet acceptance criteria . If an MNC feeder cell batch fails to meet any of the acceptance criteria outlined above, the following steps will be performed to retest the batch to rule out simple experimenter error as the cause.

If two or more satellite testing vials remain in the batch, then the batch shall be tested again. If one satellite testing vial remains in the batch, or if no satellite testing vials remain in the batch, then the batch is deemed unqualified according to the acceptance criteria listed above.

For the materials to be considered合格 (qualified), both the relevant batch and the control batch must meet the above acceptance criteria. Only after meeting these criteria is the batch permitted for use.

Example 4: Preparation of IL-2 stock solution

This embodiment describes a process for dissolving purified lyophilized recombinant human interleukin-2 in a stock sample suitable for use in other tissue culture protocols (including all those described in this application and in the embodiments), including those involving the use of rhIL-2.

Procedure. Prepare a 0.2% acetic acid solution (HAc). Transfer 29 mL of sterile water to a 50 mL conical tube. Add 1 mL of 1N acetic acid to the 50 mL conical tube. Mix thoroughly by inverting the tube 2-3 times. Sterilize the HAc solution by filtration using a Steriflip filter.

Prepare PBS containing 1% HSA. Add 4 mL of 25% HSA stock solution to 96 mL of PBS in a 150 mL sterile filter unit. Filter the solution. Store at 4°C. Complete the form for each vial of rhIL-2 prepared.

Prepare a stock solution of rhIL-2 (final concentration of 6 × ^10⁶ IU/mL). Each batch of rhIL-2 is different, and the required information can be found in the manufacturer's Certificate of Analysis (COA), such as: 1) the mass of rhIL-2 per vial (mg), 2) the specific activity of rhIL-2 (IU/mg), and 3) the recommended 0.2% HAc reduction volume (mL).

Calculate the required volume of 1% HSA for a batch of rhIL-2 using the following formula:

For example, according to the COA of rhIL-2 batch 10200121 (Cellgenix), the specific activity of a 1 mg vial is 25 × ^10⁶ IU/mg. It is recommended to reconstitute rhIL-2 in 2 mL of 0.2% HAc.

Wipe the rubber stopper of the IL-2 vial with an alcohol swab. Using a 16G needle attached to a 3mL syringe, inject the recommended volume of 0.2% HAc into the vial. Be careful not to remove the stopper while withdrawing the needle. Invert the vial three times and rotate until all powder dissolves. Carefully remove the stopper and place it on the alcohol swab. Add the calculated volume of 1% HSA to the vial.

Store rhIL-2 solution. For short-term storage (<72 hours), store vials at 4°C. For long-term storage (>72 hours), aliquot the vials into smaller volumes and store them in frozen vials at -20°C until ready for use. Avoid freeze/thaw cycles. Expiry date is 6 months after preparation date. The Rh-IL-2 label includes the supplier and catalog number, lot number, expiry date, operator initials, concentration, and aliquot volume.

Example 5: Cryopreservation Process

This embodiment describes a cryopreservation process for TILs prepared according to the procedures described herein, using a CryoMed controlled-rate cryostat (Thermo Scientific) of model 7454.

The equipment used is as follows: aluminum box holder (compatible with CS750 freezer bags), freezer box for 750mL bags, low-pressure (22psi) liquid nitrogen tank, freezer, thermocouple sensor (for the strip type of bag) and CryoStore CS750 freezer bags (OriGen Scientific).

From nucleation to -20°C, the freezing process provides a rate of 0.5°C and a cooling rate of 1°C/min until the final temperature of -80°C. The program parameters are as follows: Step 1—Wait at 4°C; Step 2: 1.0°C/min (sample temperature) to -4°C; Step 3: 20.0°C/min (chamber temperature) to -45°C; Step 4: 10.0°C/min (chamber temperature) to -10.0°C; Step 5: 0.5°C/min (chamber temperature) to -20°C; and Step 6: 1.0°C/min (sample temperature) to -80°C.

Example 6: Tumor amplification process using a defined culture medium

The procedures disclosed above can be performed using appropriate culture media (e.g., CTS ^™ OpTmizer ^™ T-cell expansion SFM, ThermoFisher, including, for example, DM1 and DM2) instead of CM1 and CM2 media.

Example 7: Exemplary GEN 2 preparation of cryopreserved TIL cell therapy

This embodiment describes cGMP manufacturing of Iovance Biotherapeutics' TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices. This embodiment describes an exemplary cGMP manufacturing of a TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices.

Table 38. Exemplary plans for process expansion.

Table 39. Culture Flask Volume

Day 0, CM1 medium preparation. In BSC, add reagents to RPMI 1640 culture bottles. Add the following reagents per bottle: heat-inactivated human AB serum (100.0 mL); GlutaMax ^™ (10.0 mL); gentamicin sulfate, 50 mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL).

Remove unnecessary materials from the BSC. Dispense culture medium reagents from the BSC, leaving gentamicin sulfate and HBSS in the BSC for use in preparing wash medium.

Thaw the IL-2 aliquot. Thaw a 1.1 mL IL-2 aliquot (6 × ^10⁶ IU/mL) (BR71424) until all ice has melted. Record the IL-2 batch number and expiration date.

Transfer the IL-2 stock solution to the culture medium. In BSC, transfer 1.0 mL of the IL-2 stock solution to the prepared CM1 Day 0 culture medium bottle. Add one bottle of CM1 Day 0 culture medium and 1.0 mL of IL-2 (6 × 10⁶ IU/mL).

Transfer G-REX100MCS to BSC. Aseptically transfer G-REX100MCS (W3013130) to BSC.

Pump all complete CM1 Day 0 culture medium into G-REX100MCS culture flasks. Alternatively, use conical tubes or G-Rex100MCS for tissue fragments.

Day 0, preparation of tumor washing medium. In BSC, 5.0 mL of gentamicin (W3009832 or W3012735) was added to a 1×500 mL HBSS medium (W3013128) bottle. Each bottle contained: HBSS (500.0 mL); gentamicin sulfate, 50 mg/mL (5.0 mL). Filtered HBSS containing gentamicin was prepared via a 1 L 0.22 μm filter unit (W1218810).

Day 0 Tumor Treatment. Obtain tumor samples and immediately transfer them to a kit at 2–8°C for treatment. Aliquot the tumor washing medium. Tumor washing 1 was performed using 8” forceps (W3009771). The tumor was removed from the sample vial and transferred to the prepared “Wash 1” culture dish. This was followed by tumor washing 2 and tumor washing 3. The tumor was measured and evaluated. It was assessed whether >30% of the total tumor area was observed to be necrotic and/or fatty tissue. Clean dissection was performed where applicable. If the tumor was large and >30% of the tissue surface was observed to be necrotic/fatty, “cleaning segmentation” was performed by removing the necrotic/fatty tissue using a combination of scalpel and/or forceps while preserving the internal structure of the tumor. The tumor was dissected. Using a combination of scalpel and/or forceps, the tumor sample was cut into an even number of appropriately sized fragments (up to 6 intermediate fragments). Intermediate tumor fragments were transferred. The tumor fragments were segmented into pieces approximately 3×3×3 mm in size. The intermediate fragments were stored to prevent dehydration. The intermediate fragment segmentation was repeated. The number of small pieces collected was determined. If only the desired tissue was preserved, additional favorable tumor fragments were selected from the “Favorable Intermediate Fragments” 6-well plate to fill the discarded fragments, so that up to 50 fragments were collected.

Prepare the conical tube. Transfer the tumor slice to a 50 mL conical tube. Prepare the BSC for G-REX100MCS. Remove the G-REX100MCS from the incubator. Aseptically transfer the G-REX100MCS culture flask to the BSC. Add the tumor fragment to the G-REX100MCS culture flask. Distribute the fragments evenly.

Incubate G-REX100MCS with the following parameters: G-REX culture flask: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Calculate: Incubation time; Lower limit = Incubation time + 252 hours; Upper limit = Incubation time + 276 hours.

After the process is complete, discard all remaining warm culture medium and thaw the aliquots of IL-2.

Day 11 - Culture medium preparation. Incubator monitoring. Incubator parameters: Temperature LED display: 37.0±2.0℃; CO2 percentage: 5.0±1.5% CO2.

Incubate 3 x 1000 mL RPMI 1640 medium (W3013112) bottles and 3 x 1000 mL AIM-V (W3009501) bottles in an incubator for ≥30 minutes. Remove the RPMI 1640 medium from the incubator. Prepare the RPMI 1640 medium. Filter the medium. Thaw 3 x 1.1 mL IL-2 aliquots (6 x 10⁶ IU/mL) (BR71424). Remove the AIM-V medium from the incubator. Add IL-2 to the AIM-V. Aseptically transfer the medium to the BSC using a 10 L Labtainer bag and relay pump transfer device.

Prepare a 10L Labtainer culture medium bag. Prepare a Baxa pump. Prepare another 10L Labtainer culture medium bag. Pump the culture medium into the 10L Labtainer. Remove the automatic pump tubing from the Labtainer bag.

Mix the culture medium. Gently knead the bag to mix. Sampling the culture medium according to the sampling plan. Take 20.0 mL of culture medium and place it in a 50 mL conical tube. Prepare the cell counting dilution tubes. In the BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" and lot number to four 15 mL conical tubes. Transfer the reagents from the BSC to 2–8°C. Prepare the 1 L transfer pack. On the outside of the BSC, fuse the 1 L transfer pack (according to process note 5.11) to the transfer device attached to the prepared "Complete CM2 Day 11 Medium" bag. Prepare the feeder cell transfer pack. Incubate the Complete CM2 Day 11 Medium.

Day 11 - TIL collection. Pretreatment form. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX100MCS from the incubator. Prepare 300mL transfer packs. Fuse the transfer packs to the G-REX100MCS.

Prepare the culture flask for TIL collection and begin the collection. Collect TILs. Using GatheRex, transfer the cell suspension through a blood filter to a 300 mL transfer pack. Examine the adherent cells on the membrane.

Rinse the culture flask membrane. Close the clamps on the G-REX100MCS. Ensure all clamps are closed. Heat-seal the TIL and "supernatant" transfer pack. Calculate the volume of the TIL suspension. Prepare the supernatant transfer pack for sampling.

Draw Bac-T samples. In the BSC, aspirate approximately 20.0 mL of supernatant from the 1 L “Supernatant” transfer pack and dispense into sterile 50 mL conical tubes.

Inoculate with BacT according to the sampling plan. Using an appropriately sized syringe, take 1.0 mL of sample from a prepared 50 mL conical tube labeled with BacT and inoculate it into an anaerobic bottle.

Incubate the TIL. Place the TIL transfer kit in an incubator only when needed. Perform cell counting and calculations. Measure the mean viable cell concentration and mean cell viability for each cell count. Viability ÷ 2. Viable cell concentration ÷ 2. Measure the upper and lower limits of the count. Lower limit: mean viable cell concentration × 0.9. Upper limit: mean viable cell concentration × 1.1. Confirm that both counts are within acceptable limits. Measure the mean viable cell concentration based on all four counts performed.

Adjusting the volume of the TIL suspension: Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume (A). Volume of the removed cell count sample (4.0 mL) (B) Adjusted total TIL cell volume C = A - B.

Calculate the total number of viable TIL cells. Average viable cell concentration*: total volume; total number of viable cells: C = A × B.

Flow cytometry calculation: If the total count of live TIL cells is ≥4.0× ^10⁷ , then calculate the volume of 1.0× ^10⁷ cells for the flow cytometry sample.

Total number of live cells required for flow cytometry: 1.0 × ^10⁷ cells. Cell volume required for flow cytometry: live cell concentration divided by 1.0 × ^10⁷ cells A.

Calculate the volume of a TIL suspension equal to 2.0 × ^10⁸ live cells. Calculate the volume of excess TIL cells to be removed as needed, and remove the excess TIL and place it in an incubator as needed. Calculate the total amount of excess TIL to be removed as needed.

The calculated volume of CS-10 medium added to the excess TIL cells at the target cell concentration for freezing is 1.0 × ^10⁸ cells/mL. Centrifuge the excess TIL as needed. Observe the conical tube and add CS-10.

Fill vials. Divide 1.0 mL of cell suspension into appropriately sized vials. Divide the remaining volume into appropriately sized vials. If the volume is ≤0.5 mL, add CS10 to the vials until the volume is 0.5 mL.

Calculate the cell volume required for 1 × ^10⁷ cells for cryopreservation. Remove the sample for cryopreservation. Place the TIL in an incubator.

Cryopreservation of samples. Observe the conical tube and slowly add CS-10, recording the volume of 0.5 mL of CS-10 added.

Day 11 - Feeder Cells. Obtain feeder cells. Obtain at least two bags of feeder cells from two different batches using an LN2 freezer. Preserve the cells on dry ice before thawing. Prepare a water bath or cryotherm. Thaw the feeder cells in a water bath or cryotherm at 37.0 ± 2.0 °C for approximately 3–5 minutes or until the ice has just melted. Remove the culture medium from the incubator. Combine the thawed feeder cells. Add the feeder cells to the transfer packs. Dispense the feeder cells from the syringe into the transfer packs. Mix the combined feeder cells and label the transfer packs.

Calculate the total volume of the feeder cell suspension in the transfer pack. Remove the cell counting samples. Using a separate 3 mL syringe, aspirate 4 × 1.0 mL of cell counting sample from the feeder cell suspension transfer pack using the non-essential injection port for each sample. Aliquot each sample into labeled cryovials. Perform cell counting, determine the multiplier, select a protocol, and enter the multiplier. Measure the mean viable cell concentration and mean cell viability for the cell count. Determine the upper and lower limits of count and confirm that they are within these limits.

Adjust the volume of the feeder cell suspension. Calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Calculate the total number of viable feeder cells. Obtain additional feeder cells as needed. Thaw additional feeder cells as needed. Place the fourth feeder cell bag in a zip-lock bag and thaw in a 37.0±2.0°C water bath or Cytotherm for approximately 3–5 minutes, and combine with the additional feeder cells. Measure the volume. Measure the volume of feeder cells in the syringe and record it below (B). Calculate the new total volume of feeder cells. Add the feeder cells to the transfer pack.

Prepare diluents as needed, adding 4.5 mL of AIM-V medium to four 15 mL conical tubes. Prepare for cell counting. Using a separate 3 mL syringe for each sample, remove 4 × 1.0 mL of the cell count sample from the feeder cell suspension transfer pack using the non-essential injection port. Perform cell counting and calculations. Determine the mean viable cell concentration based on all four counts performed. Adjust the feeder cell suspension volume and calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Subtract the removed 4.0 mL from the total feeder cell volume. Calculate the volume of feeder cell suspension required to obtain 5 x ^10⁹ viable feeder cells. Calculate the excess feeder cell volume. Calculate the volume of excess feeder cells to be removed. Remove the excess feeder cells.

Using a 1.0 mL syringe and a 16G needle, aspirate 0.15 mL of OKT3 and add OKT3. Heat-seal the feeder cell suspension transfer pack.

Day 11: G-REX Filling and Inoculation Setup for G-REX500MCS. Remove the "Complete CM2 Day 11 Medium" from the incubator and pump the medium into the G-REX500MCS. Pump 4.5L of medium into the G-REX500MCS, filling to the marked line on the culture flask. Heat seal as needed and incubate the culture flask. Fuse the feeder cell suspension transfer kit to the G-REX500MCS. Add feeder cells to the G-REX500MCS. Heat seal. Fuse the TIL suspension transfer kit to the culture flask. Add TIL to the G-REX500MCS. Heat seal. Incubate the G-REX500MCS at 37.0±2.0℃, CO2 percentage: 5.0±1.5% CO2.

Calculate the incubation window. Perform calculations to determine the appropriate time to remove the G-REX500MCS from the incubator on day 16. Lower limit: Incubation time + 108 hours. Upper limit: Incubation time + 132 hours.

Day 11: Cryopreservation of Excess TIL. Applicable to: Cryopreservation of excess TIL vials. Confirm that the Controlled Rate Freezer (CRF) has been set before freezing. Perform cryopreservation. Transfer the vial from the controlled rate freezer to the appropriate storage container. After freezing is complete, transfer the vial from the CRF to the appropriate storage container. Transfer the vial to the appropriate storage container. Record the storage location in LN2.

Day 16 Culture Medium Preparation. Preheat AIM-V medium. Calculate the warming time for culture bags 1, 2, and 3. Ensure all bags have been warmed for 12 to 24 hours. Set up the 10L Labtainer for supernatant. Attach the larger diameter end of the fluid pump transfer device to a concave port of the 10L Labtainer bag using a Luer connector. Set up and label the 10L Labtainer for supernatant. Set up the 10L Labtainer for supernatant. Ensure all clamps are closed before removing from the BSC. Note: Use the supernatant bag during TIL collection, which can be performed in parallel with culture medium preparation.

Thaw IL-2. Thaw 5 × 1.1 mL IL-2 aliquots (6 × ^10⁶ IU/mL) (BR71424) of each CTS AIM V medium bag until all ice melts. Aliquot 100.0 mL GlutaMax ^™ . Add IL-2 to GlutaMax ^™ . Prepare the CTS AIM V medium bag for preparation. Prepare the CTS AIM V medium bag for preparation. Multistage Baxa pump. Prepare the medium. Pump GlutaMax ^™ + IL-2 into the bag. Monitor parameters: Temperature LED display: 37.0 ± 2.0 °C, _CO₂ percentage: 5.0 ± 1.5% _CO₂ . Warm up the complete CM4 day 16 medium. Prepare the dilution.

Day 16, REP splitting. Incubator parameters monitored: Temperature LED display: 37.0±2.0℃, _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX500MCS from the incubator. Prepare a 1L transfer pack, label it TIL suspension, and weigh it as 1L.

The volume of the G-REX500MCS decreased. Approximately 4.5 L of culture supernatant was transferred from the G-REX500MCS to a 10 L Labtainer.

Prepare the culture flask for TIL collection. After removing the supernatant, close all clamps leading to the red tubing.

Initiate TIL collection . Vigorously tap the culture flask and rotate the culture medium to detach the cells, ensuring all cells are detached.

TIL Collection . Loosen all clamps leading to the TIL suspension transfer pack. Using GatheREX, transfer the cell suspension into the TIL suspension transfer pack. Note: Ensure the edges are kept tilted until all cells and culture medium are collected. Check for adherent cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS. Heat-seal the transfer pack containing TIL. Heat-seal the 10L Labtainer containing the supernatant. Record the weight of the transfer pack containing the cell suspension and calculate the suspension volume. Prepare the transfer pack for sample removal. Remove the test sample from the cell supernatant.

Aseptic and BacT test sampling . Take 1.0 mL of sample from the prepared 15 mL cone-labeled BacT. Take the cell counting sample. In the BSC, use a separate 3 mL syringe to take 4 × 1.0 mL cell counting samples from the TIL suspension transfer pack for each sample.

Remove the Mycoplasma sample. Using a 3 mL syringe, remove 1.0 mL from the TIL suspension transfer pack and place it into a prepared 15 mL conical tube labeled “Mycoplasma diluent”.

Prepare the transfer kit for inoculation. Place the TIL in an incubator. Remove the cell suspension from the BSC and place it in an incubator until needed. Perform cell counting and calculation. First, dilute the cell count sample to a 1:10 dilution by adding 0.5 mL of the cell suspension to 4.5 mL of prepared AIM-V medium. Measure the mean viable cell concentration and mean survival rate of the cells used for cell counting. Measure the upper and lower limits of counting. Note: Dilutions can be adjusted based on the expected cell concentration. Determine the mean viable cell concentration from all four counts performed. Adjust the volume of the TIL suspension. Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume minus the 5.0 mL removed for testing.

Calculate the total number of viable TIL cells. Calculate the total number of culture flasks to be inoculated. Note: The maximum number of G-REX500MCS culture flasks to be inoculated is five. If the calculated number of culture flasks to be inoculated exceeds five, inoculate only five culture flasks using all available volumes of cell suspension.

Calculate the number of culture flasks needed for subculture. Calculate the number of culture medium bags still needed besides the prepared bags. Prepare one 10L "CM4 Day 16 Medium" bag for every two G-REX-500M culture flasks as calculated. Continue inoculating the first G-REX-500M culture flask while preparing additional medium and warming it. Prepare the determined number of additional culture medium bags and warm them. Fill the G-REX500MCS. Prepare the pumpable medium and pump 4.5L of medium into the G-REX500MCS. Heat seal. Repeat filling. Incubate the culture flasks. Calculate the target volume of TIL suspension to be added to the new G-REX500MCS culture flasks. If the calculated number of culture flasks exceeds five, inoculate only five culture flasks using all volumes of cell suspension. Prepare the culture flasks for inoculation. Remove the G-REX500MCS from the incubator. Prepare the G-REX500MCS for pumping. Close all clamps except for the larger filter line. Remove the TIL from the incubator. Prepare the cell suspension for inoculation. Aseptically fuse the "TIL suspension" transfer package (according to process note 5.11) to the pump inlet line. Place the TIL suspension bag on the scale.

Inoculate the culture flasks with TIL suspension. Pump the calculated volume of TIL suspension into the culture flasks. Heat seal. Fill the remaining culture flasks.

Monitoring the incubator. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Incubation culture bottles.

The time range for removing G-REX500MCS from the incubator on day 22 was determined.

Day 22 Wash Buffer Preparation. Prepare a 10L Labtainer bag. Attach a 4” plasma transfer device to the 10L Labtainer bag via a Luer connector in the BSC. Prepare the 10L Labtainer bag. Close all clamps before transferring out of the BSC. Note: Prepare one 10L Labtainer bag for every two G-REX 500MCS culture flasks to be collected. Aspirate the Plasmalyte pump into the 3000mL bag and remove air from the 3000mL Origen bag by inverting the pump and manipulating the bag's position. Add 25% human albumin to the 3000mL bag. Obtain a final volume of 120.0mL of 25% human albumin.

Prepare the IL-2 diluent. Using a 10 mL syringe, remove 5.0 mL of LOVO wash buffer through the needle-free injection port on the LOVO wash buffer bag. Dispense the LOVO wash buffer into 50 mL conical tubes.

Distribute LOVO wash buffer into equal portions of the CRF blank bags. Using a 100mL syringe, draw 70.0mL of LOVO wash buffer from the needleless injection port.

Thaw one 1.1 mL IL-2 (6 × 10⁶ IU/mL) until all ice has melted. Add 50 μL of IL-2 stock solution (6 × ^10⁶ IU/mL) to a 50 mL conical tube labeled “IL-2 Diluent”.

Preparation for cryopreservation . Place 5 cryopreservation boxes at 2-8°C to pretreat them for use in the final product cryopreservation.

Prepare the cell counting dilution. In a BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" to four separate 15 mL conical tubes. Prepare for cell counting. Label the four frozen vials with vial numbers (1-4). Store the vials in the BSC for later use.

TIL collection on day 22. Monitor the incubator. Incubator parameters: Temperature LED display: 37±2.0℃, CO2 percentage: 5%±1.5%. Remove the G-REX500MCS culture flask from the incubator. Prepare and label the TIL collection bag. Seal any additional connectors. Volume reduction: Transfer approximately 4.5L of supernatant from the G-REX500MCS to the supernatant bag.

Prepare the culture flask for TIL collection. Initiate TIL collection. Vigorously tap the culture flask and rotate the culture medium to detach the cells. Ensure all cells have detached. Initiate TIL collection. Loosen all clamps leading to the TIL suspension collection bag. TIL collection. Using GatheRex, transfer the TIL suspension to a 3000 mL collection bag. Examine the adhered cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS, ensuring all clamps are closed. Transfer the cell suspension to the LOVO source bag. Close all clamps. Heat seal. Remove 4 × 1.0 mL cell counting samples.

Perform cell counting. Cell counting and calculation were performed using NC-200 and process note 5.14. The cell counting sample was first diluted by adding 0.5 mL of cell suspension to 4.5 mL of prepared AIM-V medium, resulting in a 1:10 dilution. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The upper and lower limits of counting were determined. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The LOVO source bag was weighed. The total number of viable TIL cells was calculated. The total number of nucleated cells was calculated.

Prepare the Mycoplasma diluent. Take 10.0 mL from a supernatant bag via the Luhr sample port and place it into a 15 mL conical tube.

Perform the "TIL G-REX collection" protocol and determine the target volume of the final product. Load the disposable kit. Remove the filtrate bag. Enter the filtrate volume. Place the filtrate container on the lab bench. Attach PlasmaLyte. Confirm that PlasmaLyte has been attached and that it is moving. Attach the source container to the tubing and confirm that it has been attached. Confirm that PlasmaLyte is moving.

Final preparation and filling. Target volume/bag calculation. Calculate the volume of CS-10 and LOVO wash buffer to be prepared in blank bags. Prepare CRF blank bags.

Calculate the volume of IL-2 to be added to the final product. Required final IL-2 concentration (IU/mL) - 300 IU/mL. IL-2 working stock solution: 6 × ^10⁴ IU/mL. Assemble and connect the device. Aseptically fuse the 4S-4M60 to the CC2 unit connector. Aseptically fuse the CS750 cryoprotectant bag to the prepared wire bundle. Fuse the CS-10 bag to the tip of the 4S-4M60. Prepare TILs with IL-2. Using an appropriately sized syringe, extract the measured amount of IL-2 from the “IL-2 6 × ^10⁴ ” aliquot. Label the prepared TIL bag. Add the prepared TIL bag to the device. Add CS10. Switch syringes. Aspirate approximately 10 mL of air into a 100 mL syringe and replace the 60 mL syringe on the device. Add CS10. Prepare the CS-750 bag. Dispense cells.

Remove air from the final product bag and obtain the retention. Once the last final product bag has been filled, close all clamps. Draw 10 mL of air into a new 100 mL syringe and replace the syringe on the device. Dispense the retention into 50 mL conical tubes and label the tubes “Retention” and the batch number. Repeat the air removal steps for each bag.

Prepare the final product for cryopreservation, including visual inspection. Store the cryopreservation bag on a cooling pack or at 2-8°C before cryopreservation.

Remove the cell counting sample. Using an appropriately sized pipette, remove 2.0 mL of the retained sample and place it in a 15 mL conical tube for cell counting. Perform cell counting and calculation. Note: Only dilute one sample to an appropriate confirmatory dilution. Dilute the remaining sample to the appropriate dilution factor and continue counting. Measure the mean viable cell concentration and mean viability of the cells used for cell counting. Measure the upper and lower limits of count. Note: The dilution may be adjusted according to the expected cell concentration. Measure the mean viable cell concentration and mean viability. Measure the upper and lower limits of count. Calculate IFN-γ. Heat-seal the final product bag.

The following exemplary sampling plan is used to label and collect samples.

Table 40. Sample Plan.

Sterility and BacT test . Test sampling. In the BSC, take 1.0 mL of sample from the retained cell suspension collected using an appropriately sized syringe and inoculate it into an anaerobic flask. Repeat the above steps for the aerobic flask.

Cryopreservation of the final product. Prepare the controlled-rate freezer (CRF). Verify that the CRF is set up. Set the CRF probe. Place the final product and sample in the CRF. Measure the time required to reach 4℃ ± 1.5℃ and continue the CRF operation. Complete the CRF and store. Stop the CRF after completion. Remove the box and vial from the CRF. Transfer the box and vial to gas chromatography-tandem nitrogen (LN2) for storage. Record the storage location.

The final processing and analysis of the drug included the following tests: (Day 22) CD3+ cell count of REP on Day 22 by flow cytometry; (Day 22) Gram staining method (GMP); (Day 22) bacterial endotoxin test by gel clot LAL assay (GMP); (Day 16) BacT sterility assay (GMP); (Day 16) TD-PCR (GMP) detection of Mycoplasma DNA; acceptable appearance characteristics; (Day 22) BacT sterility assay (GMP); (Day 22) IFN-γ assay. Other potency assays as described herein were also used to analyze the TIL product.

Example 8: Exemplary Implementation of the GEN 3 Amplification Platform

Day 0

Prepare tumor washing medium. Warm the medium before starting. Add 5 mL of gentamicin (50 mg/mL) to a 500 mL HBSS bottle. Add 5 mL of tumor washing medium to a 15 mL Erlenmeyer flask for OKT3 dilution. Prepare feeder cell bags. Aseptically transfer feeder cells to feeder cell bags and store at 37°C until use or freeze. If stored at 37°C, count the feeder cells. If frozen, thaw and then count the feeder cells.

The optimal range for feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with a 4.5 mL LAIM-V. Add 0.5 mL of cell fraction for each cell count. If the total number of viable feeder cells is ≥ 1 × ^10⁹ cells, adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag to add 1 × ^10⁹ cells to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of tumor washing medium to an OKT3 aliquot (100 μL). Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the second feeder cell bag. Adjust the medium volume to a total volume of 2 L. Transfer the second feeder cell bag to an incubator.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 0: 15 μg/vial, i.e., 30 ng/mL in 500 mL, up to approximately 60 μL, 1 aliquot.

Add 5 mL of tumor wash medium to all wells of a 6-well plate labeled as excess tumor flakes. Reserve the tumor wash medium for further use in maintaining tumor hydration during dissection. Add 50 mL of tumor wash medium to each 100 mm Petri dish.

Using a ruler as a reference, divide the tumor into ^27mm segments (3×3×3mm) under the dissecting culture dish lid. Dissect the intermediate segments until 60 segments are obtained. Count the total number of final segments and prepare G-REX-100MCS culture flasks (usually 60 segments/flask) based on the number of final segments produced.

Preserve favorable tissue fragments in conical tubes labeled Fragment Tubes 1 through 4. Calculate the number of G-REX-100MCS culture flasks seeded with feeder cell suspension based on the number of initial fragment tubes.

Remove the feeder cell bag from the incubator and inoculate with G-REX-100 MCS. Label it D0 (day 0).

Add the tumor fragment to the culture in the G-REX-100 MCS . Under aseptic conditions, unscrew the caps of the G-REX-100 MCS labeled "Tumor Fragment Culture (D0) 1" and the 50 mL conical tube labeled "Fragment Tube". Rotate the opened fragment tube 1 while simultaneously slightly lifting the cap of the G-REX-100 MCS. Add the culture medium and fragment together to the G-REX-100 MCS while rotating. Record the number of fragments transferred to the G-REX-100 MCS.

Once the fragment is at the bottom of the GREX culture flask, aspirate 7 mL of culture medium and create seven 1 mL aliquots: 5 mL for extended characterization and 2 mL for sterility. Store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until needed.

Inoculate one anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle with 1 mL of the final fragment culture supernatant. Repeat the above procedure for each culture bottle from which samples are taken.

On the 7th-8th day

Prepare the feeder cell bags. If frozen, thaw the feeder cell bags in a 37°C water bath for 3–5 minutes. If frozen, count the feeder cells. The optimal feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with 4.5 mL AIM-V. For each cell count, add 0.5 mL of the cell fraction to a new frozen tube. Mix the sample thoroughly and perform cell counting.

If the total number of live feeder cells is ≥2× ^10⁹ cells, proceed to the next step to adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag so that 2× ^10⁹ cells can be added to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of HBSS to 100 μL of OKT3 aliquot. Mix by pipetting three times, upwards and downwards. Prepare two aliquots.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 7/8: 30 μg/vial, i.e., 60 ng/mL in 500 mL, up to 120 μL, 2 aliquots.

Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the feeder cell bag, ensuring all is added. Adjust the culture medium volume to a total of 2 L. Repeat the above steps for the second OKT3 aliquot and add it to the feeder cell bag. Transfer the second feeder cell bag to an incubator.

Prepare G-REX100MCS culture flasks containing feeder cell suspension . Record the number of G-REX100MCS culture flasks to be processed based on the number of G-REX culture flasks produced on day 0. Remove the G-REX culture flasks from the incubator and remove the second feeder cell bag from the incubator.

Remove the supernatant before adding the feeder cell suspension . Connect a 10 mL syringe to a G-REX100 culture flask and aspirate 5 mL of culture medium. Prepare five 1 mL aliquots, 5 mL for extended characterization, and store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until requested by the initiator. Label each G-REX100 culture flask and repeat the above steps.

Depending on the number of culture flasks, 5-20 × 1 mL samples are used for characterization:

●5mL = 1 culture flask

●10mL = 2 culture flasks

●15mL = 3 culture flasks

●20mL = 4 culture flasks

Continue seeding feeder cells into G-REX100 MCS flasks, and repeat the above procedure for each G-REX100 MCS flask. Using aseptic transfer, transfer 500 mL of the second feeder cell bag by weight (assuming 1 g = 1 mL) to each G-REX-100 MCS flask and record the transfer volume. Label this as Day 7 culture and repeat the above procedure for each G-REX100 flask. Transfer the G-REX-100 MCS flasks to an incubator.

Days 10-11

Remove the first G-REX-100MCS culture flask and, under aseptic conditions, use a 10mL syringe to extract 7mL of pretreated culture supernatant. Prepare seven 1mL aliquots: 5mL for extended characterization and 2mL for sterile samples.

Carefully mix the culture flasks and use a new 10mL syringe to remove 10mL of the supernatant and transfer it to a 15mL tube labeled D10/11 Mycoplasma supernatant.

Carefully mix the culture flasks and, using a new syringe, dispense the following volumes based on the number of culture flasks to be processed:

● 1 bottle = 40mL

●2 bottles = 20mL/bottle

● 3 bottles = 13.3 mL/bottle

● 4 bottles = 10mL/bottle

Aspirate a total of 40 mL from all culture flasks and collect the samples in a 50 mL conical tube labeled ‘Day 10/11 QC Sample’. Store the tube in an incubator until needed. Perform cell counting and cell distribution.

Five aliquots (pretreated culture supernatant) used for extended characterization were stored at ≤-20°C until needed. One anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle were inoculated with 1 mL of pretreated culture supernatant, respectively.

Continue transferring the cell suspension to G-REX-500MCS and repeat the above procedure for each G-REX-100MCS. Under aseptic conditions, transfer the contents of each G-REX-100MCS to a G-REX-500MCS, monitoring approximately 100 mL of fluid transfer each time. Stop transferring when the volume of the G-REX-100MCS decreases to 500 mL.

During the transfer step, use a 10 mL syringe and aspirate 10 mL of cell suspension from the G-REX-100MCS into the syringe. Follow the instructions according to the number of flasks in culture. If only 1 flask: use two syringes to aspirate a total of 20 mL. If 2 flasks: aspirate 10 mL from each flask. If 3 flasks: aspirate 7 mL from each flask. If 4 flasks: aspirate 5 mL from each flask. Transfer the cell suspension to a common 50 mL conical tube. Keep in the incubator until the cell counting step and QC sample. The total number of cells required for QC is approximately 20e6 cells: 4 × 0.5 mL cell counts (cell counts are undiluted first).

The required cell quantity for the assay is as follows:

1. At least 10 × ^10⁶ cells should be used for efficacy assays, such as those described herein, or for IFN-γ or granzyme B assays.

2.1 × ^10⁶ cells were used for Mycoplasma.

3.5 × ^10⁶ cells were used for CD3+/CD45+ flow cytometry.

Transfer the G-REX-500MCS culture flask to an incubator.

Prepare QC samples . In this embodiment, at least 15 × ^10⁸ cells are required for the assay. Assays include: cell count and viability; Mycoplasma (1 × ^10⁶ cells/mean viable concentration); flow cytometry (5 × ^10⁶ cells/mean viable concentration); and IFN-γ assay (5 × ^10⁶ cells - 1 × ^10⁶ cells; IFN-γ assay requires 8-10 × ^10⁶ cells).

Calculate the volume of the cell fraction cryopreserved at 10 × ^10⁶ cells/mL, and calculate the number of vials required.

Days 16-17

Wash buffer preparation (1% HSA Plasmalyte A). Transfer the HSA and Plasmalyte to a 5L bag to prepare the LOVO wash buffer. Under aseptic conditions, transfer a total volume of 125 mL of 25% HSA to a 5L bag. Remove 10 mL or 40 mL of the wash buffer and transfer it to an 'IL-2 6× ^10⁴ IU/mL tube' (10 mL if the IL-2 is pre-prepared, or 40 mL if the IL-2 is freshly prepared).

Calculate the volume of restored IL-2 added to Plasmalyte + 1% HSA: Volume of restored IL-2 = (Final concentration of IL-2 × Final volume) / Specific activity of IL-2 (based on standard determination). The final concentration of IL-2 is 6 × ^10⁴ IU/mL. The final volume is 40 mL.

Take the calculated initial volume of IL-2 required for IL-2 recovery and transfer it to a tube labeled 'IL-2 6 × ^10⁴ IU/mL'. Add 100 μL of IL-2 6 × ^10⁶ IU/mL from the pre-prepared aliquot to a tube labeled 'IL-2 6 × ^10⁴ IU/mL' containing 10 mL of LOVO wash buffer.

Remove approximately 4500 mL of supernatant from the G-REX-500MCS culture flask. Rotate the remaining supernatant and transfer the cells to the cell collection pocket. Repeat the above steps for all G-REX-500MCS culture flasks.

Take 60 mL of the supernatant and add it to the supernatant tube for quality control determination, including mycoplasma detection. Store at +2–8°C.

Cell collection . Cell counting. Prepare four 15 mL conical tubes containing 4.5 mL of AIM-V. These conical tubes can be prepared in advance. Optimal range = between 5 × ^10⁴ and 5 × ^10⁶ cells/mL (1:10 dilution recommended). For a 1:10 dilution, add 500 μL of CF to the previously prepared 4500 μL of AIM-V. Record the dilution factor.

Calculate the total cells (TC) of pre-LOVO (survival + death) =

Average total cells

Concentration (pre-LOVO TC concentration)

(Survival + Death)

X

Volume of source bag

Calculate the TVC (total viable cells) of pre-LOVO (survival) =

Average total viable cells

Concentration (pre-LOVO TVC)

(Survival)

X

LOVO source bag volume

When the total cell count (TC) is >5 × ^10⁹ , remove 5 × ^10⁸ cells for cryopreservation as a reserve sample for MDA. 5 × ^10⁸ ÷ average TC concentration (step 14.44) = volume to be removed.

When the total cell count (TC) is ≤5× ^10⁹ , 4× ^10⁶ cells are removed and cryopreserved as a reserve sample for MDA. 4× ^10⁶ ÷ average TC concentration = volume to be removed.

When determining the total cell count, the number of cells to be removed should allow for the retention of 150 × ^10⁹ viable cells. Confirm the pre-LOVO TVC as 5 × ^10⁸ or 4 × ^10⁶ , or not applicable. Calculate the volume of cells to be removed.

Calculate the remaining total cells in the bag. Calculate the pre-LOVO TC (total cells). [Average total cell concentration x remaining volume = pre-LOVO remaining TC]

Select the corresponding process from Table 41 based on the total number of remaining cells.

Table 41. Total number of cells.

Select the volume of IL-2 to be added corresponding to the procedure used. The volume is calculated as follows: Residue volume × 2 × 300 IU/mL = Required IU of IL-2. Required IU of IL-2 / 6 × ^10⁴ IU/mL = Volume of IL-2 added after LOVO bagging. Record all added volumes. Obtain samples in frozen vials for further analysis.

Mix the cell products thoroughly. Seal all bags for further processing, including freezing where appropriate.

Perform endotoxin, IFN-γ, sterility, and other assays on the obtained frozen vial samples as needed.

Example 9: Exemplary Processes for GEN 2 and GEN 3

This example illustrates the Gen 2 and Gen 3 processes. The Gen 2 and Gen 3 TILs typically consist of autologous TILs derived from individual patients (via surgical tumor resection) and then expanded ex vivo. The first expansion step in initiating the Gen 3 process involves cell culture in the presence of interleukin-2 (IL-2) and the monoclonal antibody OKT3, which targets the T-cell co-receptor CD3 on the cytoskeleton of irradiated peripheral blood mononuclear cells (PBMCs).

The fabrication of the Gen 2 TIL product consists of two stages: 1) pre-rapid expansion (pre-REP) and 2) rapid expansion protocol (REP). During pre-REP, excised tumors are cut into ≤50 fragments of 2-3 mm in each dimension. These fragments are cultured for 11 days in serum-containing medium (RPMI 1640 medium supplemented with 10% HuSAB) and 6,000 IU/mL interleukin-2 (IL-2). On day 11, TILs are collected and introduced into a large-scale secondary REP expansion. The REP consists of the following: activation of ≤200 × ^10⁶ live cells from pre-REP for 5 days in ⁵ L of CM2 supplemented with 3,000 IU/mL rhIL-2, with the feeder cells loaded with 150 μg of monoclonal anti-CD3 antibody (OKT3). On day 16, the culture volume was reduced by 90%, and the cells were fractionated to ≥1× ^10⁹ viable lymphocytes/flask into multiple G-REX-500 culture flasks, and brought to a final volume of 5L with CM4. The TIL was then incubated for another 6 days. On day 22, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

The fabrication of Gen 3 TIL products consists of three stages: 1) initiation of the first expansion protocol, 2) rapid second expansion protocol (also known as the rapid expansion phase or REP), and 3) dissociation of subcultures. To achieve initiation of the first expansion TIL proliferation, the excised tumor was fragmented into ≤120 fragments, each 2-3 mm in dimensions. On day 0 of initiation of the first expansion, a feeder layer of approximately 2.5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was established on a surface area of approximately 100 ^cm² in each of three 100 MCS dishes. The tumor fragments were distributed in the three 100 MCS dishes and cultured for 7 days in CM1 medium containing 500 mL of serum and 6,000 IU/mL of interleukin-2 (IL-2) and 15 μg of OKT-3. On day 7, REP was initiated as follows: an additional feeder cell layer of approximately 5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was incorporated into the tumor fragmentation culture phase in each of three 100 MCS dishes and cultured with 500 mL CM2 medium and 6,000 IU/mL IL-2 and 30 μg OKT-3. REP initiation was enhanced by activating the entire initiation first expansion culture in the same dish, which was achieved by transferring OKT3-loaded feeder cells to the 100 MCS dishes using a closed-system fluid transfer. For Gen 3, TIL scaling up or splitting involved the following procedural steps: the entire cell culture was scaled up and transferred to the larger dish via a closed-system fluid transfer (from a 100 M culture flask to a 500 M culture flask) and an additional 4 L CM4 medium was added. On day 16, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

Overall, the Gen 3 process is a shorter, more scalable, and easier-to-modify amplification platform that will be suitable for stable manufacturing and process comparability.

Table 50. Comparison of the manufacturing processes of Exemplary Gen 2 and Exemplary Gen 3.

On day 0, for both processes, tumor cells were washed three times and fragments were randomly grouped into two pools; one pool for each process. For the Gen 2 process, fragments were transferred to a GREX 100MCS flask containing 1 LCM1 of medium with 6,000 IU/mL rhIL-2. For the Gen 3 process, fragments were transferred to a G-REX100MCS culture flask containing 500 mL CM1 of medium with 6,000 IU/mL rhIL-2, 15 μg OKT-3, and 2.5 × ^10⁸ feeder cells. TILs were inoculated on Rep start day according to different days for each process. For the Gen 2 process, where the volume of the G-REX-100MCS culture flasks decreased by 90%, the collected cell suspension was transferred to new G-REX-500MCS on day 11 to initiate REP in CM2 medium containing IL-2 (3000 IU/mL) plus 5 × ^10⁹ feeder cells and OKT-3 (30 ng/mL). Cells were expanded according to the protocol and split into multiple G-REX-500MCS culture flasks containing CM4 medium and IL-2 (3000 IU/mL) on day 16. Cultures were then collected and cryopreserved on day 22 according to the protocol. For the Gen 3 process, REP initiation was performed on day 7 using the same G-REX-100MCS. In short, 500 mL of CM2 medium containing IL-2 (6000 IU/mL), 5 × ^10⁸ feeder cells, and 30 μg OKT-3 was added to each culture flask. On days 9–11, scale up the culture. Transfer the entire volume of G-REX100M (1 L) to G-REX-500MCS and add 4 L of CM4 containing IL-2 (3000 IU/mL). Incubate the flask for 5 days. On day 16, collect the culture and freeze it.

The comparison included three different tumors: two lung tumors (L4054 and L4055) and one melanoma tumor (M1085T).

For L4054 and L4055, CM1 (medium 1), CM2 (medium 2), and CM4 (medium 4) media were prepared in advance and maintained at 4°C. CM1 and CM2 media were prepared without filtration to compare cell growth with and without filtration of the media.

For L4055 tumors, the culture medium was heated at 37°C for up to 24 hours during REP initiation and scaling up.

Results . For the total number of viable cells achieved, the results for Gen 3 were within 30% of those for Gen 2. Upon restimulation, the Gen 3 final product exhibited higher IFN-γ production. As measured by the total number of unique CDR3 sequences present, the Gen 3 final product showed increased clonal diversity. The Gen 3 final product also exhibited a longer mean telomere length.

Pre-REP and REP expansion in Gen 2 and Gen 3 processes followed the procedures described above. For each tumor, both pools contained an equal number of fragments. Due to the smaller size of the tumors, the maximum number of fragments per flask could not be reached. Total pre-REP cells (TVCs) were collected and counted on day 11 of the Gen 2 process and day 7 of the Gen 3 process. To compare the two pre-REP groups, the cell count was divided by the number of fragments provided in the culture to calculate the average number of viable cells per fragment. As indicated in Table 51 below, more cells were consistently grown per fragment in the Gen 2 process compared to the Gen 3 process. The extrapolation of the expected TVC number on day 11 of the Gen 3 process was calculated by dividing the pre-REP TVC by 7 and then multiplying by 11.

Table 51. Pre-REP cell count

*L4055, unfiltered culture medium.

For both Gen 2 and Gen 3 processes, TVCs were counted according to process conditions, and the percentage of viable cells per day of the process was generated. At harvest, cells were collected on day 22 (Gen 2) and day 16 (Gen 3), and TVC counts were generated. The TVCs were then divided by the number of fragments provided on day 0 to calculate the average number of viable cells per fragment. The fold increase was calculated by dividing the collected TVCs by the REP initiation TVCs. As shown in Table 52, comparing Gen 2 and Gen 3, the fold increases were similar for L4054; in the case of L4055, the fold increase was higher for the Gen 2 process. Specifically, in this case, the medium was heated to 24°C prior to the REP initiation day. A higher fold increase was also observed in Gen 3 for M1085T. The extrapolation of the expected TVC count on day 22 of the Gen 3 process was calculated by dividing the REP TVCs by 16 and then multiplying by 22.

Table 52. Total viable cell count and fold expansion of TIL final products.

*L4055, unfiltered culture medium.

Table 53: Survival percentage of final TIL products: Final TIL REP products were compared to the survival percentage release criteria after collection. All conditions for Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Following collection, the final TIL REP product was compared against release criteria based on survival percentage. All conditions for both Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Table 53. Survival Percentage of REP (TIL Final Product)

Because the number of fragments in each vial is less than the maximum required number, the estimated cell count on the collection day is calculated for each tumor. This assessment is based on the expectation that the clinical tumor is large enough on day 0 to be inoculated with 2 or 3 vials.

Table 54. Extrapolation of estimated cell counts to full-size 2 and 3 bottles for the Gen 3 process.

Immunophenotypic Analysis – Comparison of Phenotypic Markers in TIL End Products. Three tumor types, L4054, L4055, and M1085T, underwent TIL amplification during both Gen 2 and Gen 3 processes. Following collection, REP TIL end products were analyzed by flow cytometry to test for purity, differentiation, and memory markers. For all conditions, the percentage of TCR a/b+ cells exceeded 90%.

TILs collected from the Gen 3 process showed higher expression of CD8 and CD28 compared to those collected from the Gen 2 process. The Gen 2 process showed a higher percentage of CD4+.

TILs collected from the Gen 3 process showed higher expression of the central memory chamber compared to TILs collected from the Gen 2 process.

Activation and depletion biomarkers were analyzed in TILs from two tumors, L4054 and L4055, to compare the final TIL products from the Gen 2 and Gen 3 TIL amplification processes. Activation and depletion biomarkers for the Gen 2 and Gen 3 processes were similar.

Interferon-γ secretion upon restimulation. On the collection days, day 22 of Gen 2 and day 16 of Gen 3, TILs of L4054 and L4055 were restimulated overnight using coated anti-CD3 plates. M1085T was restimulated using anti-CD3, CD28, and CD137 beads. Under all conditions, the supernatant was collected and frozen 24 hours after restimulation. IFNγ analysis by ELISA was performed simultaneously on the supernatants from both processes using the same ELISA plate. Higher IFNγ production from the Gen 3 process was observed in all three tumors analyzed.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption during Gen 2 versus Gen 3 processes, cell supernatants were collected on REP initiation, scale-up, and collection days for tumors L4054 and L4055. The amount of IL-2 in the cell culture supernatant was measured using a Quantitate ELISA kit from R&D. A general trend indicates that IL-2 concentrations remained higher during Gen 3 compared to Gen 2. This may be attributed to the higher IL-2 concentration at Gen 3 REP initiation (6000 IU/mL) and residual culture medium throughout the process.

Metabolic substrate and metabolite analysis. The levels of metabolic substrates (e.g., D-glucose and L-glutamine) are measured as substitutes for overall culture medium consumption. Their reciprocal metabolites, such as lactate and ammonia, are also measured. Glucose is a monosaccharide in the culture medium, used by mitochondria to produce energy in the form of ATP. When glucose is oxidized, lactate is produced (lactate is an ester of lactate). Lactate is produced in large quantities during the exponential growth phase of cells. High levels of lactate can negatively impact cell culture processes.

For Gen 2 and Gen 3 processes, used culture media from L4054 and L4055 were collected on REP start, scale-up, and collection days. Used culture media were collected on days 11, 16, and 22 of Gen 2, and on days 7, 11, and 16 of Gen 3. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

L-Glutamine is an unstable essential amino acid required in cell culture media formulations. Glutamine contains an amine, and this amide group allows for the transport and delivery of nitrogen into cells. When L-glutamine is oxidized, cells produce toxic ammonia byproducts. To counteract L-glutamine degradation, GlutaMax ^™ was supplemented into the culture medium during the Gen 2 and Gen 3 processes, as it is more stable in aqueous solution and does not spontaneously degrade. In both tumor lines, the Gen 3 group showed a decrease in L-glutamine and GlutaMax ^™ during the process, as well as an increase in ammonia throughout the REP. In the Gen 2 group, constant L-glutamine and GlutaMax ^™ concentrations were observed, along with a slight increase in ammonia production. For ammonia, the Gen 2 and Gen 3 processes were similar at the collection day, showing only slight differences in L-glutamine degradation.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats at L4054 and L4055 was measured using Flow-FISH technology during Gen 2 and Gen 3. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Gen 3 showed similar telomere lengths to Gen 2.

CD3 analysis. To determine the clonal diversity of cell products generated in each process, the collected TIL final products from L4054 and L4055 were sampled and analyzed by sequencing the CDR3 portion of the T cell receptor for clonal diversity analysis.

Table 55 shows a comparison of the percentage of unique CDR3 sequences shared on L4054 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 199 sequences with Gen 2 final products, and 97.07% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 55. Comparison of the common uCDR3 sequence between Gen 2 and Gen 3 processes on L4054.

Table 56 shows a comparison of the percentage of unique CDR3 sequences shared on L4055 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 1833 sequences with Gen 2 final products, and 99.45% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 56. Comparison of common uCDR3 sequences between Gen 2 and Gen 3 processes on L4055.

CM1 and CM2 culture media were prepared in advance without filtration and kept at 4°C until used for tumor L4055 for Gen 2 and Gen 3 processes.

For L4055 tumors, on the REP initiation day, the culture medium was heated at 37°C for 24 hours for use in the Gen 2 and Gen 3 processes.

LDH was not measured in the supernatant collected during the process.

M1085T TIL cells were counted using a K2 cellometer.

On the tumor M1085T, samples are unavailable, such as supernatant for metabolic analysis; TIL products for activation and depletion marker analysis, telomere length and CD3-TCR vb analysis.

Conclusion . This example compares three independent donor tumor tissues based on functional quality attributes, expanded phenotypic characterization of the Gen 2 and Gen 3 processes, and culture medium consumption.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

Assuming pre-REP collection is performed on day 11 instead of day 7 and REP collection is performed on day 22 instead of day 16, extrapolated cell numbers for the Gen 3 process were calculated. In both cases, Gen 3 showed closer TVC numbers compared to the Gen 2 process, indicating that early activation enhances TIL growth.

Extrapolating values from other flasks (2 or 3) in the Gen 3 process, assuming larger tumor sizes and the maximum number of fragments required for each process as described. It was observed that collections performed on day 16 of the Gen 3 process achieved similar TVC doses compared to day 22 of the Gen 2 process. This observation is significant and indicates that early activation of the culture may reduce TIL treatment time.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

In terms of phenotypic characterization, higher CD8+ and CD28+ expression was observed in all three tumors during the Gen 3 process compared to the Gen 2 process.

Compared to the Gen 2 process, the Gen 3 process shows a slightly higher central memory compartment.

Although the Gen 3 process is shorter in duration, the Gen 2 and Gen 3 processes show similar activation and exhaustion markers.

In the three tumors analyzed, the IFNγ production on the final product of Gen 3 was 3-fold higher than that of Gen 2. This data suggests that the Gen 3 process produces a more potent and efficient TIL product compared to the Gen 2 process, which may be attributed to higher expression of CD8 and CD28 on Gen 3. Phenotypic characterization showed a positive trend in CD8+ and CD28+ expression in Gen 3 compared to the Gen 2 process in all three tumors.

The telomere lengths of the final TIL products from Gen 2 and Gen 3 are similar.

The glucose and lactate levels of the final products of Gen 2 and Gen 3 were similar, indicating that the nutrient levels on the culture medium were not affected during the Gen 3 process because no reduction removal was performed each day during the process and the overall culture medium volume was smaller compared to Gen 2.

Compared to the Gen 2 process, the processing time of the entire Gen 3 process is reduced by about half, which will significantly reduce the cost of goods sold (COG) of TIL products amplified through the Gen 3 process.

IL-2 consumption indicates the general trend of IL-2 consumption during the Gen 2 process, and that IL-2 is higher during the Gen 3 process due to the lack of removal of old culture medium.

CDR3 TCRab sequence analysis revealed high clonal diversity in the Gen 3 process.

Adding feeder cells and OKT-3 on day 0 of the pre-REP allows for early activation of TILs and enables TIL growth using the Gen 3 process.

Table 57 describes the various implementation schemes and results of the Gen 3 process compared to the current Gen 2 process.

Table 57. Exemplary Gen 3 process characteristics.

Example 10: Exemplary GEN 3 process (also known as GEN 3.1)

This embodiment describes further studies regarding the comparability between Gen 2 and Gen 3 processes for TIL amplification. The Gen 3 process is modified to include an activation step early in the process, thereby increasing the final total live cells (TVC) output while maintaining phenotypic and functional profile. As described below, the Gen 3 implementation is modified to another implementation, referred to herein as Gen 3.1.

In some implementations, the Gen 3.1 TIL manufacturing process involves four operator interventions:

1. Tumor Fragment Isolation and Activation: On day 0 of the procedure, the tumor was dissected and final fragments of approximately 3 × ³ mm were generated (up to 240 fragments in total) and cultured in 1–4 G-REX100MCS culture flasks. Each flask contained up to 60 fragments, 500 mL of CM1 or DM1 medium, supplemented with 6,000 IU rhIL-2, 15 μg OKT3, and 2.5 × 10⁸ irradiated allogeneic mononuclear cells. The cultures were incubated at 37°C for 6–8 days.

2. TIL Culture Reactivation: On days 7-8, in both cases, replenish the culture by slowly adding CM2 or DM1 medium supplemented with 6,000 IU rhIL-2, 30 μg OKT3, and 5 × ^10⁸ irradiated allogeneic monocytes. Take care not to disturb the existing cells at the bottom of the culture flask. Incubate the culture at 37°C for 3-4 days.

3. Culture scaling up: Performed on days 10-11. During culture scaling up, in both cases, transfer all contents of the G-REX100MCS to a G-REX500MCS culture flask containing 4 L of CM4 or DM2 supplemented with 3,000 IU/mL IL-2. Incubate the culture flask at 37°C for 5-6 days until harvest.

4. Collection/Washing/Preparation: On days 16-17, reduce the volume of the culture flasks and combine them. Concentrate the cells and wash them with PlasmaLyte A pH 7.4 containing 1% HSA. Prepare the washed cell suspension with CryoStor10 at a 1:1 ratio and supplement with rhIL-2 to a final concentration of 300 IU/mL.

DPs are cryopreserved and stored in gaseous liquid nitrogen via controlled-rate freezing. *Complete standard TIL media 1, 2, or 4 (CM1, CM2, CM4) can be used instead of CTS ^™ OpTmizer ^™ T cell serum-free expansion medium, also known as the defining medium (DM1 or DM2), as mentioned above.

Procedure Description. On day 0, the tumor was washed three times and then fragmented into 3×3×3 final fragments. After fragmenting the entire tumor, the final fragments were then equally randomized and divided into three pools. One randomized fragment pool was introduced into each group, with the same number of fragments added according to the three experimental matrices.

Throughout the TIL amplification process, tumor L4063 was amplified using standard culture medium, and tumor L4064 was amplified using a specific culture medium (CTS OpTmizer). The components of the culture media are described herein.

CM1 complete medium 1: RPMI + glutamine, supplemented with 2mM GlutaMax ^™ , 10% human AB serum, gentamicin (50ug/mL), and 2-mercaptoethanol (55uM). The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM2 complete medium 2: 50% CM1 medium + 50% AIM-V medium. The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM4 complete medium 4: supplemented with AIM-V containing GlutaMax ^™ (2mM). The final medium formulation was supplemented with 3000 IU/mL IL-2.

The basal ^{medium for} T cell expansion is supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L).

DM1: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 6,000 IU/mL IL-2.

DM2: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 3,000 IU/mL IL-2.

All types of culture media used, i.e., complete (CM) and defined (DM) media, were prepared in advance and kept at 4°C until the day before use, and were preheated in an incubator at 37°C for up to 24 hours prior to the treatment day.

For both tumor types, TIL cultures were reactivated on day 7. For L4063, reactivation was performed on day 10, and for L4064, scale-up was performed on day 11. Both cultures were collected and cryopreserved on day 16.

Results achieved . Cell counts and survival percentages were determined during Gen 3.0 and Gen 3.1 procedures. Amplification under all conditions followed the details described in this example.

For each tumor, the fragments were divided into three equal pools. Due to the small size of the tumors, the maximum number of fragments per bottle could not be reached. Total viable cells and cell viability were assessed under each condition for three different procedures. Cell counts were determined as TVC at reactivation on day 7, TVC at scaling up on day 10 (L4064) or day 11 (L4063), and TVC at collection on day 16/17.

Cell counts on days 7 and 10/11 were considered as FIO. Folds of expansion were calculated by dividing the TVC at day 16/17 (when collected) by the TVC at day 7 (reactivation). To compare the three groups, the TVC at the collection day was divided by the number of fragments added to the culture on day 0 to calculate the average number of viable cells per fragment.

Cell counting and viability assays were performed on L4063 and L4064. On both tumors, the Gen3.1 assay produced more cells per fragment than the Gen 3.0 assay.

Total viable cell count and fold expansion; percentage of survival during the procedure. Percentage of survival under all conditions was obtained after reactivation, scaling up, and collection. Release criteria for survival percentage were compared to final TILs after collection on day 16/17. All conditions assessed exceeded the 70% survival criterion and were similar across all procedures and tumors.

Immunophenotyping analysis – Phenotypic characterization of TIL final products. Flow cytometry analysis of the final products was performed to test purity, differentiation, and memory markers. Under all conditions, the population percentages of TCRα/β, CD4+, and CD8+ cells were constant.

Extended phenotypic analysis of REP TILs was performed. In both tumors, TIL products showed a higher percentage of CD4+ cells under Gen 3.1 conditions compared to Gen 3.0, and a higher percentage of CD28+ cells from the CD8+ population under Gen 3.0 conditions compared to Gen 3.1 conditions under both conditions.

TILs collected from Gen 3.0 and Gen 3.1 processes showed phenotypic markers similar to CD27 and CD56 expression on CD4+ and CD8+ cells, and similar CD28 expression on CD4+-gated cell populations. Comparison of memory markers on TIL final products:

Frozen samples of TIL collected on day 16 were stained for analysis. The TIL memory state was similar in the Gen 3.0 and Gen 3.1 processes. Comparison of activation and depletion markers on the final TIL products:

The activation and depletion markers of gated Gen 3.0 and Gen 3.1 processes are similar in CD4+ and CD8+ cells.

Interferon-γ secretion after restimulation. For L4063 and L4064, collected TILs were restimulated overnight using coated anti-CD3 plates. In both tumors analyzed, higher IFNγ production from the Gen 3.1 process was observed compared to the Gen 3.0 process.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption levels under all conditions and processes, cell supernatants were collected and frozen at the start of reactivation on day 7, scale-up on day 10 (L4064)/day 11 (L4063), and collection on day 16/day 17. The supernatants were then thawed and analyzed. The amount of IL-2 in the cell culture supernatant was measured according to the manufacturer's protocol.

IL-2 consumption was similar throughout the entire Gen 3 and Gen 3.1 processes, evaluated under the same culture conditions. IL-2 concentrations (pg/mL) were analyzed in the used culture media collected from L4063 and L4064.

Metabolite analysis. For each condition, used culture supernatant was collected from L4063 and L4064 at day 7 for reactivation initiation, day 10 (L4064)/day 11 (L4063) for scaling up, and day 16/day 17 for collection. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

Compared to the complete medium (2 g/L), the glucose concentration in the determined medium was higher, at 4.5 g/L. Overall, the glucose concentration and consumption were similar for the Gen 3.0 and Gen 3.1 processes across each medium type.

An increase in lactate was observed, and the increase in lactate was similar under Gen 3.0 and Gen 3.1 conditions as well as in the two media (complete medium and deterministic medium) used for reactivation amplification.

In some cases, the standard basal medium contains 2 mM L-glutamine and is supplemented with 2 mM GlutaMax ^™ to compensate for the natural degradation of L-glutamine into L-glutamic acid and ammonia under culture conditions.

In some cases, the specified (serum-free) culture medium used does not contain L-glutamine compared to the basal medium and is supplemented only with GlutaMax ^™ at a final concentration of 2 mM. GlutaMax ^™ is a dipeptide of L-alanine and L-glutamine, which is more stable than L-glutamine in aqueous solution and does not spontaneously degrade into glutamate and ammonia. Instead, the dipeptide gradually dissociates into individual amino acids, thereby maintaining a low but sufficient concentration of L-glutamine to sustain stable cell growth.

In some cases, the concentrations of glutamine and GlutaMax ^™ decreased slightly on scale-up days but increased on collection days, reaching levels similar to or closer to those on reactivation days. For L4064, the concentrations of glutamine and GlutaMax ^™ showed a slight decrease at a similar rate under different conditions throughout the process.

The ammonia concentration was higher in the samples grown in the standard medium containing 2 mM glutamine + 2 mM GlutaMax ^™ compared to the samples grown in the defined medium containing 2 mM GlutaMax ^™ . Furthermore, as expected, a gradual increase or accumulation of ammonia was observed during cultivation. No difference in ammonia concentration was observed under the three different test conditions.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats on L4063 and L4064 cells during the Gen 3 and Gen 3.1 processes was measured using Flow-FISH technology. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Telomere measurements were performed. Telomere lengths in the samples were compared with those in the control cell line (1301 leukemia). The control cell line was a tetraploid cell line with long, stable telomeres that allowed for the calculation of relative telomere lengths. Similar telomere lengths were observed in the Gen 3 and Gen 3.1 processes evaluated in both tumors.

TCR Vβ lineage analysis

To determine the clonal diversity of cell products generated in each process, TIL end products were analyzed for clonal diversity analysis by sequencing the CDR3 portion of the T cell receptor.

Compare three parameters across three conditions:

● Diversity index of unique CDR3 (uCDR3)

● Total uCDR3 percentage

●For the first 80% of uCDR3:

○ Compare the total percentage of uCDR3 copies

○ Compare the frequency of unique clones

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 975 sequences, which is equivalent to 88% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 2163 sequences, which is equivalent to 87% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

The number of unique CD3 sequences identified from 1 × ^10⁶ cells collected on day 16 for different processes. Based on the number of unique peptide CDRs within the sample, the Gen 3.1 test conditions showed slightly higher clonal diversity compared to Gen 3.0.

The Shannon entropy diversity index is a reliable and commonly used comparative measure because the Gen 3.1 condition showed slightly higher diversity in both tumors compared to the Gen 3 process, indicating that the TCRVβ lineage of the Gen 3.1 test condition was more polyclonal than that of the Gen 3.0 process.

Furthermore, for tumors L4063 and L4064, the TCR Vβ lineage under the Gen 3.1 test conditions showed more than 87% overlap with the corresponding lineage of the Gen 3.0 process.

On the reactivation day, the IL-2 concentration on the used medium for the Gen 3.1 test of L4064 was lower than expected (similar to the Gen 3.1 control and Gen 3.0 conditions).

The low value may be attributed to pipetting error, but since the sample size was very small, it was impossible to repeat the measurement.

Conclusion . Compared with the Gen 3.0 and Gen 3.1 controls, the Gen 3.1 test conditions, which included feeder cells and OKT-3 on day 0, showed a higher TVC at day 16 collection. The TVC of the final product under the Gen 3.1 test conditions was approximately 2.5 times higher than that under Gen 3.0.

For the two tumor samples tested, the Gen 3.1 test conditions, with the addition of OKT-3 and feeder cells on day 0, reached the maximum capacity of the culture flasks at collection. Under these conditions, if a maximum of four flasks were started on day 0, the final cell dose could be between 80 and 100 × ^10⁹ TILs.

Maintain all quality attributes, such as phenotypic characterization, including the purity, depletion, activation, and memory markers of the final TIL product, between the Gen 3.1 test and the Gen 3.0 process.

In both tumor types analyzed, the final TIL product IFN-γ production in Gen 3.1 with added feeder cells and OKT-3 on day 0 was 3-fold higher than that in Gen 3.0, indicating that the Gen 3.1 process produces efficient TIL products.

No differences in glucose or lactate levels were observed under any of the test conditions. No differences in glutamine and ammonia were observed between the Gen 3.0 and Gen 3.1 processes under any of the culture medium conditions. Lower glutamine levels in the culture medium did not limit cell growth, indicating that the addition of GlutaMax ^™ alone to the culture medium is sufficient to provide the nutrients required for cell proliferation.

Scaling up was performed on days 11 and 10, respectively, and no significant difference was observed in the number of cells reached on the process's collection day. Furthermore, metabolite consumption was similar throughout the process in both cases. These observations suggest that the Gen 3.0 optimized process offers flexibility in terms of processing days, thereby promoting flexibility in manufacturing schedules.

As measured by CDR3 TCRab sequence analysis, the Gen 3.1 process, which added feeder cells and OKT-3 on day 0, showed higher clonal diversity compared to Gen 3.0.

Figure 32 illustrates the implementation scheme of the Gen 3 process (Gen 3 optimization process). Standard medium and serum-free CTS Optimizer medium can be used for TIL amplification in the Gen 3 optimization process. When using serum-free CTS Optimizer medium, it is recommended to increase the final concentration of GlutaMax ^™ on the medium to 4 mM.

Example 11: PD-1 gene knockout in TIL using TALEN

The embodiment described above illustrates the use of TALEN to generate PD-1 gene knockout (KO) TILs. In this embodiment, the general procedure is as follows:

Day 0 - Pre-REP

Day 7 - Activation

Day 12 - Electroporation

Day 13 - REP

Day 18 - Scale Expansion

Day 22 - Collection

Overview : This study used genetic engineering techniques to permanently knock out the PDCD-1 gene (PD-1 surface protein) using an mRNA construct. PD-1KO TILs were generated from one lung (L4256) and two melanoma (M1207 and M1209) tumors and then characterized.

Background : Binding between immune checkpoint receptors and their ligands has been shown to suppress T cell function. PD-1, a key inhibitory regulator expressed by activated/depleted T cells, has been extensively studied and is a major target for immunotherapy. Given the success of anti-PD-1 therapy in improving clinical outcomes in cancer patients, approaches targeting the PD-1/PD-L1 axis could enhance the efficiency of immunotherapy. Adoptive TIL therapy is a salvage immunotherapy for cancer, with an objective response rate of 56% in melanoma patients. Since PD-1 is regulated in TILs in response to antigen stimulation in vivo, the interaction between PD-1 and PD-L1 expressed by tumor cells may suppress TIL function. Gene editing of PD-1 has demonstrated high specificity for permanent gene disruption in human and mouse T cells. In the B16 mouse model, adoptive transfer of PD-1 T cells revealed superior tumor control and enhanced effector function and proliferative capacity of tumor-site infiltrating T cells. Therefore, this study examines whether PD-1 KO-mediated TILs can enhance TIL function in vivo after adoptive transfer.

Experimental design : Clinical manufacturing scale operations were performed as shown in the experimental layout in Figure 34, and an overview of the clinical manufacturing PD-1KO TIL process is provided in Tables 42 and 43.

The method is also applicable to the generation of PD-1/CTLA-4 dual-gene knockout TILs, PD-1/LAG-3 dual-gene knockout TILs, PD-1/CISH dual-gene knockout TILs, PD-1/TIGIT dual-gene knockout TILs, and PD-1/CBL-B dual-gene knockout TILs. In such cases, during the electroporation step, dual-gene knockout TILs can be generated by electroporating TILs with PD-1 and CTLA-4 TALEN mRNA, PD-1 and LAG-3 TALEN mRNA, PD-1 and CISH TALEN mRNA, PD-1 and TIGIT TALEN mRNA, or PD-1 and CBL-B TALEN mRNA, respectively.

Table 42: Outline of Pre-REP, Activation, Electroporation, and Resting Processes (Part I)

Table 43: Outline of REP, Scale-up, and Collection Process (Part II)

Results : Table 44 specifies the acceptance criteria used to evaluate the full-scale experimental efficacy, Table 45 specifies the additional end-product characterization tests performed, and Table 46 lists the tumors and related histories used in this study.

Table 44: In-process and Collected Product Release Testing and Acceptance Standards

Table 45: Characterization of the final product

Table 46: Tumors used in this study

On day 5, the total live cell count (TVC) after pre-REP yielded an average of 45 × ^10⁶ cells, with a mean survival rate of 73%. Cell doubling after activation was consistent across all three tumor types. Melanoma and the cervical indication both showed similar activation status, with activation markers of >50% CD25+ and CD71+. However, the lung tumor L4213 showed lower CD71 expression, at 33%. Ki67 expression was consistently greater than 90% in all three samples, indicating active cell proliferation. Table 47 summarizes the pre-REP and post-activation output for the three tumor types used in the study.

Table 47: Summary of Pre-REP and Post-Activation Cell Culture

parameter	Acceptance Standards	L4256	M1207	M1209
					Number of segments	≤100 segments	73	40	95
<![CDATA[TVC(×10<sup>6</sup>)]]>	≥5	26.9	13.4	69.2
					Survival rate (%)	N/A	97.9	88.6	97.5
<![CDATA[TVC(×10<sup>6</sup>)]]>	N/A	120.0	37.8	419.0
					Survival rate (%)	N/A	92.0	82.6	92.8
Double (D7 to D12)	N/A	2.2	1.5	2.6
					CD45+ / CD3+%	N/A	80.4	83.1	60.1
<![CDATA[CD25+%<sup>1</sup>]]>	N/A	92.2	88.5	66.8
					<![CDATA[CD71+%<sup>1</sup>]]>	N/A	75.6	87.8	48.9
<![CDATA[Ki-67+%<sup>2</sup>]]>	N/A	78.6	81.0	7.3

^1. Gating strategy of the group after activation on day 10: Lymphocytes > SSC single peak > FSC single peak > Live cells > CD3+CD45 > CD25+/CD71+

^2. Ki67 group's gating strategy: Lymphocytes > SSC unimodal > FSC unimodal > Live cells > CD45+ > Ki67+

On day 13 (after rest), survival rate and TVC recovery rate were calculated to determine the efficiency of electroporation. For all conditions, the recovery rate of the percentage of cells surviving after electroporation ranged from 82% to 92%. In electroporated samples, the TVC recovery rate ranged from 16% to 30%. Data from electroporation and resting are summarized in Table 48.

Table 48: Summary of Electroporation and REP Initiation

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The recovery rate is calculated by dividing the TVC or survival rate after electroporation by the TVC or survival rate before electroporation and then multiplying by 100.

^2. The TVCs injected into the REP were the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

The same number of cells were used to initiate the REP under both experimental and control conditions. As the REP scaled up, the average cell doubling occurred 4 times under all experimental and control conditions. At REP collection, TVC yield in the TriLink experimental group was similar to that in the simulated control group (TVC difference ≤20%). Non-electroporated TVC yield was slightly higher than that in the TriLink and simulated control groups. An average of 9.8 cell doublings was observed between day 11 and day 20.

The final product doses of the TriLink group were 16.5, 28.5, and 28.7 × ^10⁹ , respectively, with >90% cell viability and >98% CD45+CD3+ cell count. The final product was a highly enriched TIL product.

The data from the REP initiation and collection are summarized in Table 49.

Table 49: Post-REP Summary

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The TVC inoculated into the REP was the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

^2. The recovery rate is calculated by dividing the TVC after LOVO by the TVC before LOVO and multiplying by 100.

^3. Assuming all cells proceed to the hypothetical yield in both experimental and control conditions. On day 12 (before electroporation), cells are allocated for experimental (TriLink mRNA) and control conditions (simulated). Hypothetical yield is calculated by multiplying the collection by 3.

^4. Cell doubling is calculated using the formula “＝LOG(TVC on day 22 / TVC on day 11) /LOG(2)”.

^5. LOVO did not follow the plan.

^{The six} batches of materials were small in scale and did not undergo LOVO testing.

All TriLink mRNAs met the acceptance criteria.

Data on the gene knockout efficiency of PD-1KO TIL products evaluated are summarized in Table 50.

Table 50: TIL PD-1KO Summary

Data evaluating the TIL function of the collected samples (including IFNg, granzyme-B, and CD107a release output) are summarized in Table 51.

Table 51: Summary of TIL Functions

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. Stimulation gating targeting live cells/CD3+

The functionality of TILs was characterized based on overnight stimulation of the final product with anti-CD3/anti-CD28/anti-CD137 Dynabeads. Supernatants were collected and frozen 24 hours after stimulation. ELISA was performed to assess the concentrations of IFNg and granzyme B released into the supernatant. IFNg release was greater than 500 pg/mL, and all TIL cultures secreted high levels (15036 pg/mL–40185 pg/mL) of granzyme B after stimulation.

CD107a (LAMP-1) is a marker of lymphocyte degranulation and is associated with cytotoxic activity following antigen or polyclonal stimulation (Aktas E et al., 2009). Flow cytometry was used to analyze the cell surface expression of CD107a in response to PMA stimulation for 2 hours in both CD4 ⁺ and CD8 ⁺ cells. The results presented in Table 51 compare CD107A expression measurements with those under unstimulated conditions. Similar to the TIL products generated in previous studies, most of the TILs in the final product expressed CD107A upon stimulation with PMA/IO (CD4+ and CD8+ TILs).

Multicolor flow cytometry was used to characterize TIL purity, identity, and the memory subset of REP TILs. ≤1% of detectable B cells, monocytes, or NK cells were present in the final collected TILs (Table 11). REP TILs were primarily composed of TCRa/b cells with effector memory differentiation. The CD4/CD8 ratio varied between samples but was consistent across different conditions within a single sample. No significant differences were observed between experimental and control conditions.

Data showing TIL purity, identity, and memory phenotype are summarized in Table 52.

Table 52: Characterization of TIL purity, identity, and memory phenotype

Note: The gating strategy for TIL purity is as follows:

Monocytes: viable cells%, CD14+

NK (Natural Killer) cells: viable percentage, CD14-, CD3-, CD56+CD16+

B cells: viable percentage, CD14-, CD3-, CD19+

T-CM-Central Memory T Cells

T-EM-effect memory T cells

T-EFF/TEMRA-effect memory and RA+ effect memory

Data showing TIL activation and TIL depletion levels for CD4+, CD8+, and CD3+ are summarized in Tables 53-55.

Table 53: Activation and Depletion States of CD4+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD4+ > CD4+ frequency %

Table 54: Activation and Exhaustion States of CD8+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD8+ > CD8+ frequency %

The CD4 and CD8 differentiation, activation, and depletion states under the TriLink mRNA condition were comparable to those under the control condition.

Table 55: Activation and Depletion States of CD3+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD3+ frequency %

The activation and depletion status of TriLink mRNA in the CD3+ population were comparable to those in the control population.

Conclusion : The PD-1KO TIL process was developed at clinical manufacturing scale to amplify PD-1KO TIL to >16e9 within 20 days. All three batches of TriLink mRNA used at development scale met the acceptance criteria for release parameters.

All quality properties of TILs generated using the PD-1-KO TIL process, such as phenotypic characterization (including purity, memory, activation, exhaustion markers, and function), are comparable to those of melanoma Gen 2. See Table 56.

In summary, this work demonstrates that greater than 50% PD-1KO efficiency can be achieved in the clinical manufacturing of PD-1KO TILs using TriLink mRNA, thus supporting the use of TriLink mRNA in clinical manufacturing.

Table 56: Summary Table

The alternative PD-1 gene knockout (KO) TIL process is shown in Figure 35 and Table 57.

Table 57: Overview of the alternative full-scale process for PD-1 TIL culture

Example 12: Preclinical activity and manufacturing feasibility of gene-modified PDCD-1 gene knockout (KO) tumor-infiltrating lymphocyte (TIL) cell therapy

background

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TILs; lifileucel, LN-145) has demonstrated encouraging efficacy and safety in both ICI-injected and ICI-naïve settings in patients with advanced solid tumors. A single-dose lifileucel TIL cell therapy achieved a durable response in a post-ICI setting in patients with advanced/unresectable melanoma (Sarnaik AA, Hamid A, Khushalani NI et al., J Clin Oncol. 2021; 39(24):2656-2666; Larkin JMG, Sarnaik A, Chesney JA et al., J Clin Oncol. 2021; 39(Supplement 15): Abstract 9505.), with an objective response rate (ORR) of 36%, and the duration of response (DOR) was not reached after 33.1 months of follow-up. In patients with advanced melanoma who have not received ICI treatment, early combination therapy with lifileucel and pembrolizumab resulted in an ORR of 60% and a complete response rate of 30% (O'Malley D, Lee S, Psyrri A et al., J Immunother Cancer. 2021; 9(Supplement 2): Abstract 492).

Despite the effectiveness of anti-programmed cell death protein (PD)-1 ICI therapy, it is limited by poor tumor penetration, internalization, and endocytic clearance (Thurber GM, Schmidt MM, Dane Wittrup K. Adv Drug Deliv Rev. 2008; 60(12):1421-1434; Less JR, Posner MC, Boucher Y, Borochovitz D, Wolmark N, Jain RK. Cancer Res. 1992; 52(22):6371-6374; Netti PA, Baxter LT, Boucher Y, Skalak R, Jain RK. Cancer Res. 1995: 55(22): 5451-5458; Bordeau BM, Balthasar JP. Cancer Biol Med. 2021; 18(3): 649-664; Saga T, Neumann RD, Heya T et al., Proc Natl Acad Sci U SA. 1995; 92(19): 8999-9003); In contrast, TILs are actively transported into the tumor, can move independently by chemitraction, and are not cleared or internalized (Restifo NP, Dudley ME, Rosenberg SA. Nat Rev Immunol. 2018; 12(4): 269-281). Administration of anti-PD-1 antibodies is associated with immune-related adverse events (AEs) due to nonspecific upregulation of immune pathways via systemic activation and proliferation of T cells (Postow MA, Sildow R, Hellmann MD. N Engl J Med. 2018; 378:158-168).

IOV-4001 is a PDCD-1 gene knockout (KO) TIL cell therapy that enhances the efficacy of TIL cell therapy and eliminates the need for systemic anti-PD-1 therapy, while avoiding short-term and long-term systemic adverse events associated with anti-PD-1/PD-L1 therapy. Transcription activator-like effector endonucleases (TALENs) are hybrid molecules composed of a DNA-binding domain and a FokI nuclease. Combination of the two TALEN arms of the PDCD-1 gene encoding PD-1 mediates DNA double-strand breaks, leading to gene damage and PD-1 inactivation (Gautron AS, Juillerat A, Guyot V et al., Mol Ther Nucleic Acids. 2017; 9:312-321; Menger L, Sledzinska A, Bergerhoff K et al., Cancer Res. 2016; 76:2087-2093; Qasim W, Zhan H, Samarasinghe S et al., Sci Transl Med. 2017; 9(374):eaaj2013). A process has been established to generate TALEN-mediated PDCD-1KO TILs and amplify them to a treatment-relevant number with potent effector function and phenotypic markers indicating functional TILs (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; Sep 19–21, 2020. Abstract 1052p).

This embodiment describes the (1) preclinical activity of IOV-4001 in vivo, (2) clinical-scale manufacturing process development, and (3) phenotypic and functional characterization.

method

In summary, hIL-2NOG mice transplanted with melanoma tumor cells received autologous PD-1KO TILs (generated using an mRNA construct), TIL-mimicking (electroporation without TALEN), TIL-mimicking plus anti-PD-1 antibody adoptive transfer, or no adoptive transfer (n=14 each). Tumor size was measured twice weekly for 39 days. A 22-day clinical-scale manufacturing process was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, for the generation of PD-1KO TILs. The final PD-1KO TIL product was characterized by total live cells (TVC), purity (survival percentage), identity (CD45 ⁺ CD3 ⁺ %), and function (PD-1KO efficiency).

manufacture :

A 22-day clinical-scale manufacturing process for IOV-4001 was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, to generate PDCD-1KO TILs (using an mRNA construct). Development operations were performed at Iovance Process Development (Tampa, FL) on a non-Good Manufacturing Practice (GMP) scale. Manufacturing operations were performed at a contract manufacturing organization (CMO) on a GMP scale.

In vivo antitumor activity :

Mice expressing human interleukin-2 (hIL2) under the control of the cytomegalovirus promoter (hIL-2NOG)15 were transplanted with melanoma tumor cells and received (A) autologous PDCD-1KO TIL, (B) TIL mimicry (TIL electroporation without TALEN), (C) TIL mimicry plus anti-PD-1 antibody, or (D) TIL-free adoptive transfer (n=14 each). Tumor size was measured twice a week for 39 days.

Product release :

The final PDCD-1KO TIL product was characterized as follows:

●Total viable cells (TVC) and purity (percentage of viable cells) were determined using an NC-200 ^™ (ChemoMetec, Denmark) automated cell counterstaining system via acridine orange/4',6-diamidindo-2-phenylindole (DAPI) counterstaining.

● Identity (CD45+CD3+ phenotype), analyzed by immunofluorescence staining and flow cytometry.

●Efficacy (interferon-γ [IFNγ] release), measured by ELISA using an IFNγ ELISA kit (R&D Systems, Minneapolis, MN, USA).

PDCD-1KO efficiency was evaluated by flow cytometry based on PD-1 expression in PDCD-1KO TILs compared with mimic TILs.

Phenotype :

Two multicolor flow cytometry teams were used to determine extended phenotypic markers of the final collected PDCD-1KO TIL products to characterize TIL purity, identity, memory subsets, activation, and depletion states.

Characterization :

IL-2 assay: To assess the safety of the PDCD-1KO TIL product, the in vitro proliferation capacity of the final product in the absence of IL-2 was evaluated over a 28-day period.

Karyotype determination: Cytogenetic examination was performed by NeoGenomics Laboratories (Fort Myers, FL, USA). Briefly, cryopreserved PDCD-1KO TIL samples were quiescent and activated to collect metaphase cells for G-banding cytogenetic analysis. Three replicates of mitotic cells were analyzed, fixed, and stained for G-banding.

Statistical analysis :

Unpaired Student's t-test was used to analyze phenotypic differences, and Wilcoxon's rank-sum test was used to detect differences in in vivo studies in mice; p < 0.05 was considered statistically significant.

result

In summary, for mice treated with PD-1KO TIL (6±2.8), mean ± SEM tumor size ( ^mm² ) at day 39 showed superior tumor control compared to simulated TIL (26±8.5, P<0.05), simulated TIL + anti-PD-1 (33±8.8, P<0.01), and no adoptive TIL metastasis (112±8.4, P<0.0001). Product properties from six clinical-scale manufacturing operations of PD-1KO TIL were acceptable, with median (range) TVC, purity, and identity of 8.3 × ^10⁹ (0.9 × ^10⁹ –35.7 × ^10⁹ ), 94% (91%–99%), and 99% (98%–99%), respectively. The median (range) PD-1KO efficiency was 48% (31%–84%). PD-1KO TILs exhibit comparable functions and phenotypes (differentiation, memory, activation, and exhaustion) to those of analog TILs.

As shown in Figure 38, enhanced in vivo antitumor activity was observed in mice treated with PDCD-1KO TIL compared to mice treated with TIL-mimicking alone or TIL-mimicking alone plus anti-PD-1 antibody.

As shown in Figure 39, all PDCD-1KO TIL products met the release criteria for dose, purity, identity, potency, and PDCD-1KO efficiency. No statistically significant differences were observed between the development and manufacturing processes. Both development and manufacturing processes produced final PDCD-1KO TIL products with comparable doses (A) and survival rates (B). The median (range) identity (CD45+CD3+%) of the final TIL products in the development and manufacturing processes was 98.5% (98%–100%) and 98.7% (96%–99%), respectively (C). The median IFNγ release in the development and manufacturing processes of the final PDCD-1KO TIL products was 4015 pg/mL and 4725 pg/mL, respectively (D). The median (range) PDCD-1KO efficiencies in the development and manufacturing processes were 63% (48%–81%) and 62% (31%–91%), respectively (E). The PDCD-1KO TIL product is comparable to the simulated TIL in terms of growth, purity, identity, and potency. As previously shown (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; September 19–21, 2020. Abstract 1052p), the dose, purity, identity, and potency results between the simulated and PDCD-1KO were comparable in development operations (data not shown).

As shown in Figure 40, the CD28 biomarker was highly expressed in both PDCD-1KO and simulated TILs in the development and manufacturing samples, while other biomarkers such as CD27, CD57, and KLRG1 were expressed at low levels (A). Subsets of naive (TN), central memory (TCM), effector memory (TEM), and effector memory RA+ (TEMRA) T cells were defined using CD45RA and CCR7 expression. Most TIL batches primarily exhibited the effector memory phenotype (B). No statistically significant differences in TIL differentiation biomarkers or memory phenotypes were observed between PDCD-1KO and simulated TILs during the development and manufacturing operations.

As shown in Figure 41, multicolor flow cytometry was used to characterize TIL activation and inhibitory receptor expression on CD4+ (A) and CD8+ TILs (B). No statistically significant differences in biomarker expression were observed between PDCD-1KO and simulated TILs during development and manufacturing operations.

As shown in Figure 42, the IL-2-independent proliferation assay demonstrated that no PDCD-1KO TIL products underwent malignant transformation after TALEN-mediated genome editing. In the absence of IL-2 (A and B), neither stimulated (anti-CD3/CD28) nor unstimulated samples proliferated. All samples cultured in the presence of IL-2 showed proliferation (A and B).

The karyotype analysis results from the PDCD-1KO TIL products are summarized in Figure 43. No clonal chromosomal abnormalities were observed in the G-banding analysis, indicating no genotoxicity following TALEN-mediated PDCD-1 genome editing. All samples analyzed by G-banding generated sufficient metaphase cells for a comprehensive study; normal G-banding patterns were observed.

in conclusion

In the presence or absence of anti-PD-1, PDCD-1KO TILs exhibited superior in vivo antitumor activity compared to mimic TILs (electroporation without TALEN), indicating that endogenous PD-1 inhibition can confer a functional advantage over antibody combinations. Clinical-scale manufacturing of PDCD-1KO TILs is feasible, and the quality properties and phenotype of the TIL products are acceptable. TIL properties were comparable across all development and manufacturing operations and met product release criteria. As determined by IL-2-independent proliferation assays, no malignant transformation occurred in PDCD-1KO TIL products following TALEN-mediated genome editing. No TALEN-induced clonal chromosomal abnormalities were identified by G-banding. Importantly, the absence of intact PDCD-1KO in the TIL products preserved other PD-1-dependent in vivo cellular functions. In conclusion, these data support the clinical investigation of IOV-4001 (an autologous PDCD-1KO TIL cell therapy) as a potential treatment option for patients with advanced solid tumors.

Example 13: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated, electroporated, and subjected to an 11-day REP process.

Pre-REP cells were stimulated for two days with plate-bound OKT3 (300 ng/ml) or TransAct (1:100), followed by electroporation of 5e6 cells resuspended in an electroporation buffer called “T buffer” in 4 mm spaced electroporation reaction cuvettes, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

After electroporation, cells were allowed to rest overnight at 30°C in CM1 medium containing IL-2, followed by REP. Simulated cells were seeded at different initial numbers (100kJ, 50kJ, 20kJ, 10kJ) and tests were performed to compare gene knockout efficiency.

result

Figures 44A and 44B show cell viability and recovery folds before electroporation. Specifically, Figure 44A shows cell viability after thawing compared to 2 days after stimulation with OKT3 or TransAct, and Figure 44B shows the recovery folds after thawing and stimulation with OKT3 or TransAct.

Figures 45A and 45B show the cell viability and recovery fold of cells after electroporation. Specifically, Figure 45A shows the recovery fold of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C, and Figure 45B shows the cell viability of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C.

Figures 46A-46C show that, in the case of CD3+, CD8+, and CD4+ cells, TILs stimulated by TransAct exhibited lower KO efficiency compared to TILs stimulated by OKT3.

Example 14: Comparison of overnight rest at 30°C and 37°C

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated and electroporated.

Pre-REP cells were stimulated with TransAct (1:100) for two days, followed by electroporation of 5e6 cells resuspended in T buffer in electroporation reaction cups with a 4 mm spacing, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

Following electroporation, cells were seeded in equal numbers into 96-well plates and allowed to rest overnight at 30°C or 37°C in CM1 medium containing IL-2 or IL-15. Cells were then counted to determine the fold resuscitation and viability.

result

Figures 47A and 47B show cell viability and recovery folds after electroporation under all conditions (IL-2 or IL-15). Specifically, Figure 47A shows the recovery folds of cells after overnight rest at 30°C or 37°C, and Figure 47B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 48A and 48B show the cell viability and fold recovery rate of cells after electroporation when 6000 IU/mL IL-2 was used. Specifically, Figure 48A shows the fold recovery rate of cells after overnight rest at 30°C or 37°C, and Figure 48B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 49A and 49B show cell viability and recovery folds after electroporation under various conditions. Specifically, Figure 49A shows the recovery folds of cells after overnight rest at 30°C or 37°C when using various concentrations of IL-2 or IL-15, and Figure 49B shows the cell viability after overnight rest at 30°C or 37°C when using different concentrations of IL-2 or IL-15.

Gene knockout efficiency was evaluated as described in Example 13. TILs were stimulated overnight with anti-CD3/CD28 beads at a ratio of 1 bead to 5 cells. Figures 50A-50C show that a decrease in KO efficiency was observed at the recommended 1:100 dilution under non-GMP TransAct stimulation.

Example 15: Comparison of TALEN mRNA concentration and incubation conditions

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed, activated, electroporated and subjected to a 10-day REP process.

Pre-REP cells were stimulated with OKT3 (300 ng/mL plate-bound) for two days, followed by electroporation of 1e6 cells resuspended in T buffer in electroporation reaction cuvettes with a 1 mm spacing. The right arm or left arm was given 4 μg, 2 μg, 1 μg, and 0.5 μg of PD-1 TALEN per 1e6 cells, respectively.

After electroporation, cells were seeded in the same number in 48-well plates and allowed to rest overnight in CM1 medium containing IL-2 at 30°C or 37°C, followed by REP.

result

Figures 51 and 52 show the cell viability and fold recovery rate of cells after electroporation with different concentrations of PD-1 TALEN mRNA. Specifically, Figure 51 shows the cell viability after electroporation with different concentrations of PD-1 TALEN mRNA, and Figure 52 shows the fold recovery rate after electroporation with different concentrations of PD-1 TALEN mRNA. No significant changes in viability or recovery rate were observed after electroporation with different concentrations of PD-1 TALEN mRNA.

Figures 53A and 53B show the cell viability and fold recovery rate when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C. Specifically, Figure 53A shows the fold recovery rate of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C, and Figure 53B shows the cell viability of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C. No significant changes in viability or recovery rate were observed when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C.

Gene knockout efficiency was evaluated as described in Example 13. Figures 54A-54C show the PD-1 KO efficiency in CD3+, CD8+, and CD4+ cells. The optimal KO efficiency of PD-1 TALEN was observed at 4 μg mRNA/million cells and overnight rest at 30°C. This concentration can be target-specific and also depends on the cell concentration used during electroporation. The concentration of TALEN mRNA used during the electroporation process did not appear to affect recovery rate and survival.

Example 16: Comparison of washing steps and centrifugation speed

method

Pre-REP TIL lines (N=4) from different indications (head and neck and lung) were thawed, activated (OKT3 plate binding 300 ng/mL), and subjected to washing steps that are usually performed before electroporation.

Pre-REP cells were stimulated for two days and then aliquoted into individual tubes at 5e6 cells per tube. They were washed with PBS, precipitated, washed with cell perforation buffer, precipitated again, and then resuspended in cell perforation buffer. Cells were resuspended in 500 μL of cell perforation buffer and counted by diluting the cells 1:10 in CM1. Cell counting was performed after each rotation to determine the percentage of cells lost during the washing step.

Repeat the procedure using different centrifugation speeds (400g, 300g, 200g, for 5 minutes) to improve cell recovery rate.

result

Figures 55A and 55B show the cell number (Figure 55A) and viability (Figure 55B) after various washing steps involving centrifugation at 400g for 5 minutes and rotation 3 times.

Figures 56A and 56B show the number of cells after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 57A and 57B show cell viability after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 58A and 58B show the total number of cells and the total survival rate of cells after various rotation conditions, and Figure 59 shows the percentage of cells lost after various rotation conditions.

The results showed that washing with cell perforation buffer led to cell loss, and that the recovery rate may have been improved when cells were centrifuged in cell perforation buffer at 300g instead of 400g. Therefore, a single cell perforation buffer washing step is preferred.

Example 17: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates containing 300 ng/mL, TransAct 1:10 and TransAct 1:17.5, and then electroporated with PD-1 TAL.

result

Figures 60A-60C show the percentage loss and survival during electroporation using various stimulation methods, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation (Figure 60C). The results indicate that TransAct stimulation exhibits better recovery and survival rates after electroporation compared to OKT3.

Figures 61A-61C show the PD-1 gene knockout efficiency in CD3+ (Figure 61A), CD4+ (Figure 61B), and CD8+ (Figure 61C) cells using various stimulation methods. The results indicate that activation with OKT3 and TransAct leads to similar KO efficiency.

Figures 62A-62B show the cell viability (Figure 62A) and fold expansion (Figure 62B) of REP collections using various stimulation methods.

Example 18: Comparison of incubation temperatures after electroporation

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates at 300 ng/mL, and then electroporated with PD-1 TAL.

After electroporation, cells were incubated overnight at different temperatures (25, 28, 30, 32, 35, 37 °C) and REP was performed the following day. Cell loss and viability, as well as PD-1 gene knockout efficiency, were evaluated after overnight incubation.

result

Figures 63A-63B show the percentage of cell loss (Figure 63A) and cell viability (Figure 63B) after electroporation at different incubation temperatures.

Figures 64A-64C show the gene knockout efficiency in CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells using different incubation temperatures.

Figures 65A-65B show the fold expansion (Figure 65A) and cell viability (Figure 65B) of REP collections at different incubation temperatures.

Example 19: Comparison of Stimulus Day Timing Selection

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated with GMP TransAct 1:17.5 on different dates (days 0, 3, 5, and 7).

Electroporation of cells with PD-1 TAL was performed on days 9 and 12 to determine the optimal dates for stimulating pre-REP cells to maximize PD-1 KO efficiency and pre-REP cell number.

result

Figures 66A-66C show cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C) of cells stimulated on different dates.

Figure 67 shows the percentage growth of cells stimulated on different dates at 3 days of rest.

Example 20: Overview of PD-1KO TIL Products

"PD-1KO TIL Product" is an autologous tumor-infiltrating lymphocyte (TIL) preparation. These lymphocytes have been genome-edited with a transcription activator-like effector nuclease (TALEN) to disrupt the gene for programmed cell death protein-1 (PD-1). The PD-1 TALEN is engineered to bind to a distinct DNA sequence adjacent to the nucleotide lysis target site on the PDCD1 gene. DNA binding leads to the dimerization of two separate FokI endonuclease domains, producing a functional DNA endonuclease that can generate a sequence-specific double-strand break (DSB) in exon 2 (the central protein-coding region of PDCD1). This DSB is repaired via endogenous error-prone DNA repair pathways (e.g., non-homologous end joining (NHEJ)), resulting in nucleotide insertions or deletions, thereby disrupting the protein-coding sequence of PD-1.

The 4-step PD-1KO TIL product manufacturing process is a modified version of the method used to produce the autologous TIL product lifileucel (IND 16317 and 16819). This process consists of the following steps: a first step of TIL growth in the presence of IL-2, followed by transient activation of T cells with TransAct (a bead-free colloidal polymer nanomatrix conjugated with humanized CD3 and CD28 agonists), electroporation of PD-1KO TALEN mRNA (left and right arms), overnight rest at 30°C, and a final rapid expansion protocol (REP) step.

Cross-study PD-1 KO TIL product characterization data (Table 58) and process development data indicate that changes in the manufacturing process do not affect product quality (characteristics, survival rate, PD-1 gene knockout efficiency) and result in an average PD-1 KO efficiency of approximately 60%. In the PDX mouse model system, its antitumor activity is superior to that of the simulated control TIL in the presence of pembrolizumab.

Table 58. List of Non-Clinical Studies

Abbreviations: GLP = Good Laboratory Practice; IL-2 = Interleukin-2; KO = Gene Knockout; OCA = Oligonucleotide Capture Assay; PD-1 = Programmed Cell Death Protein-1; PDCD1 = Gene of Programmed Cell Death Protein-1; PDX = Patient-Derived Xenograft; TALEN = Transcription Activator-like Effector Nuclease; TCR = T Cell Receptor; TIL = Tumor-Infiltrating Lymphocytes

¹ This study evaluates different batches of PD-1KO TIL products, some of which were evaluated as part of a GLP compliance study.

Overview

The non-clinical studies conducted support the safety of the PD-1KO TIL product initiating clinical studies through the following:

● Confirmed that PD-1 TALEN expression in TILs is transient after electroporation;

● Characterization using NGS confirmed that 19 of the top 20 potential off-target sites identified in the OCA analysis were free of TALEN-related mutagenesis. Minimal off-target activity was detected at one candidate site (Cand 3). This is unlikely to pose a safety risk to the product because of the low mutation frequency and the fact that the site corresponds to a non-coding RNA locus not known to be associated with the disease.

● No clonal chromosomal aberrations were identified in the karyotype analysis of the PD-1KO TIL drug product, therefore there is no evidence that TALEN-mediated genome editing has significant genotoxicity;

● No evidence of IL-2-independent proliferation was found, thus demonstrating that no malignant transformation was observed in any of the PD-1KO TIL drug products tested.

Furthermore, non-clinical studies support the potential antitumor activity and mechanism of action of PD-1KO TIL products based on the following observations:

● Reproducible high levels of TALEN-mediated PDCD1 mutagenesis (approximately 60% on average) were achieved in the genome of autologous TILs, with the PDCD1 mutagenesis rate correlated with decreased PD-1 expression. Therefore, PD-1KO TIL products constitute true PD-1KO gene therapy products.

● The general characteristics of autologous TILs, which are considered crucial for anti-tumor activity, have been shown to be unaffected or positively affected by PD-1KO, including survival, proliferative capacity, differentiation status, memory T cell subset, activation/exhaustion status, TCR Vβ diversity, T cell effector function, and cell lysis activity.

● Enhanced tumor burden control was observed in PDX mouse models treated with ACT and PD-1KO TIL products, compared with control groups that did not use ACT, ACT with unmodified autologous TIL, and ACT with unmodified autologous TIL combined with anti-PD-1 antibody.

In summary, these studies demonstrate that isolating, expanding, and genetically engineering PD-1 knockout T cells from various tumors is safe and exhibits potent antitumor activity in both in vitro and in vivo models. These observations suggest that PD-1KOTIL products possess the potential and manageable safety profile of an autoimmune therapy for treating patient populations constituting unmet medical needs.

result

Figures 68A-68C show the PD-1 gene knockout efficiency. Figure 68A shows the PD-1 KO efficiency determined by measuring PD-1 receptor expression on the PD-1 KO TIL product using flow cytometry. Figure 68B shows the PD-1 KO efficiency determined by measuring the total number of PDCD1 genes with insertions/deletions using NGS. Figure 68C shows the correlation between the PD-1 KO efficiency assessed by flow cytometry and the PDCD1 gene modifications determined using NGS.

Figure 69 shows that most of the insertions/deletions in PDCD1 after TALEN genome editing were nucleotide deletions.

Phenotypic characterization

The proliferative capacity, survival, and immunophenotype of PD-1KO TIL products relative to unmodified autologous TILs were evaluated using a study-scale process derived from five different types of tumors (breast (n=5), lung (n=2), HNSCC (n=2), melanoma (n=2), and ovary (n=1)).

Cell viability and proliferation capacity

The PD-1KO TIL product exhibited cell viability comparable to non-electroporated (NE) TILs, with an average viability of approximately 93% for both types of samples. The proliferative capacity of the PD-1KO TIL product was also comparable to that of NE TILs, with average fold increases of 1217 and 1565, respectively, during the REP step of the production process.

Differentiation status of PD-1KO TIL products and NE TIL

PD-1KO TILs exhibit a differentiation state comparable to NE TILs, as determined by similar expression levels of CD27, CD28, CD56, BTLA, and KLRG-1. Therefore, PD-1 inactivation does not alter the differentiation state of TILs.

Memory T cell subsets in PD-1KO TIL products and NE TIL

In this study, over 98% of PD-1KO TIL products and NE TILs were T effector cells (CD45RA-CCR7-), while _{the TCM} subset (CD45RA-CCR7+) accounted for less than 1% of the cells analyzed. Importantly, the proportion of terminally differentiated T cells was negligible.

Activation and depletion states in PD-1KO TIL products and NE TIL

As indicated by the expression levels of CD69, CD25, and DNAM, the activation state of the PD-1KO TIL product was relatively high and comparable to that of NE TIL. Consistent with its recent activation, both the PD-1KO TIL product and NE TIL expressed high levels of co-inhibitory molecules (TIGIT and TIM-3). These results suggest that the activation and depletion states are not altered by PD-1 inactivation.

In summary, PD-1 gene knockout did not significantly affect TIL survival, proliferation capacity, or immune phenotype, indicating that the activity of PD-1KO TIL products associated with these biological characteristics was similar to that observed in unmodified autologous TILs.

Figures 70A-70B show the distribution of TCR Vβ isotypes in the CD3+PD-1- subset within the main PD-1KO TIL products and NE TILs. Main TILs generated from lung tumor L4022 (Figure 70A) and breast tumor EP11017 (Figure 70B) were electroporated with PD-1 TALEN mRNA (PD-1KO) or not electroporated (NE). Twenty-four TCR Vβ families were evaluated by flow cytometry in the CD3+PD-1-T cell subset. The percentage of TCR Vβ isotypes is plotted in pie charts. All remaining isotypes not identified by flow cytometry are pooled and shown in light blue (other). The color represents each TCR Vβ isotype, as indicated in the legend below the pie chart.

Figures 71A-71B show the effector function of PD-1KO TILs as measured by MLR and multifunctionality. For Figure 71A, TILs were generated in duplicate from melanoma (n=3), breast cancer (n=1), and lung cancer (n=1) samples. The responsiveness of NE TILs (black circles) and PD-1KO TILs (white circles) to HLA-mismatched lymphocytes was tested. IFN-γ secretion from the supernatant was assessed using ELISA. The horizontal and vertical bars represent the mean and standard error, respectively. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance. For Figure 71B, TILs were generated from melanoma (n=1) and breast cancer (n=3) samples. The multifunctionality intensity index (PSI), a measure of the ability to produce multiple cytokines at the single-cell level, was measured in activated host PD-1KO and NE TILs. The results are shown as a bar graph of the mean and corresponding standard error of cytokines involved in the inflammatory (pink), regulatory (beige), chemoattractive (purple), stimulative (blue), and effector (green) pathways. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance.

In vivo studies: Patient-derived xenograft (PDX) mouse model studies

This study compared the antitumor activity of PD-1KO TIL products with unmodified TILs in a patient-derived xenograft (PDX) model grown in hIL-2 transgenic mice. Paired autologous PDX tumor cell lines/TIL formulations derived from freshly excised human melanoma lesions (n=1; M1152) were used in this study. The PD-1KO efficiency level was 75%. No differences in cell viability, proliferation, differentiation, memory T cell subsets, or exhaustion status were observed between M1152 PD-1KO and simulated TILs.

Characterization of PDX-derived melanoma tumor cell lines

The M1152 PDX tumor cell line is a PDX tumor-derived gene from NSG mice implanted with melanoma tumor fragments. The PDX melanoma cell line was characterized by measuring melanoma tumor-specific markers called melanoma-associated chondroitin sulfate proteoglycan (MCSP) and PD-L1 expression via flow cytometry. Approximately 80% of the tumor cells expressed MCSP protein, indicating that the majority of these cells were of melanoma origin. IFN-γ treatment induced an upregulation of PD-L1 from 13% to 82% within 48 hours. This encapsulates an in vivo drug resistance mechanism in which tumor cells increase PD-L1 expression in response to T cell secretion of IFN-γ during tumor cell lysis and supports the relevance of the M1152 PDX model for assessing M1152 PD-1 KO TIL activity.

In vivo tumor burden control of M1152 PD-1KO TIL

To determine the effect of PD-1KO TILs on tumor growth, PD-1KO or TIL-mimicking cells were adoptively transferred into hIL-2NOG mice implanted with autologous melanoma cells. Combinations of anti-PD-1 and TIL-mimicking cells served as controls for PD-1/PD-L1 pathway blockade. The in vivo efficacy of PD-1KO TILs was assessed by tumor burden reduction.

Figure 72 shows the in vivo antitumor activity of the M1152 PD-1KO TIL product. hIL-2NOG mice (n=14 per treatment group) transplanted with melanoma tumor cells underwent adoptive metastasis with either PD-1KO or a TIL-mimicking agent. Anti-PD-1 antibody treatment in combination with a TIL-mimicking agent was included as a control for PD-1/PD-L1 blockade. Tumor volume was calculated as length × width and plotted as a function of time post-implantation. Statistical significance was assessed using the Wilcoxon rank-sum test, with *, **, and **** indicating p-values <0.05, <0.01, and <0.0001, respectively, and considered statistically significant.

Tumor reduction was observed in mice treated with PD-1KO and TIL-mimicking antibodies, with or without anti-PD-1 antibody therapy. This indicates that TILs can recognize and lyse transplanted tumor cells, thus validating the tumor-specific recognition of TILs via MHC and TCR conjugation. Furthermore, even in combination with anti-PD-1 therapy, PD-1KO TILs induced superior tumor control relative to TIL-mimicking TILs. The failure of anti-PD-1 to enhance the in vivo efficacy of TIL-mimicking TILs in immunodeficiency and the lack of primary and secondary lymphoid organs in hIL-2NOG mice suggests that the in vivo mechanism of systemic PD-1 suppression may require an intact immune system. Conversely, the improved antitumor activity of PD-1KO TILs compared to TIL-mimicking TILs indicates that endogenous PD-1 suppression confers a functional advantage on TILs.

Therefore, this study demonstrates that TALEN-mediated PD-1KO confers a functional advantage on TILs. This finding is consistent with several published studies that describe an enhanced ability of PD-1KO T cells to control tumor growth relative to unmodified T cells (Menger 2016, Marotte 2020).

Figures 73A-73B show the persistence of TALEN protein in autologous TILs as a function of time, as measured by Western blotting. TILs generated from melanoma (n=6; M1011, M1017, M1025, M1030, M1034, M1040) were electroporated with PD-1 TALEN mRNA. NE TILs were used as a baseline control. TALEN protein was detected in whole-cell extracts from TILs by Western blotting at 8, 10, 12, 14, and 18 hours post-electroporation (Figure 73A) and on days 1, 2, 4, and 6 (Figure 73B). The intensity of the TALEN-specific bands was quantified by densitometric analysis, with the resulting OD values averaged over time and background corrected for the mean values of NE TILs. The mean values are plotted as bars, and the standard error is shown as a vertical line.

Quantification of mutagenesis at target sites and candidate off-target sites in PD-1 TALEN

Information about each candidate off-target site is included in Table 59.

Table 59. Off-target site analysis

IL-2-independent proliferation assay

By day 28 of analysis, none of the PD-1KO TIL product samples evaluated using CD3/CD28 activation proliferated in the absence of IL-2. All samples cultured in the presence of IL-2 proliferated by day 28, with a minimum amplification of 1.7-fold and a maximum of 407-fold. Notably, paired TALEN-edited TILs and unmodified TILs isolated from the same tumor showed comparable overall amplification folds across samples. These results demonstrate the safety of the PD-1KO TIL drug product by confirming the absence of cell proliferation in the absence of IL-2, indicating no malignant transformation of cells in this product.

Cytogenetics/karyotype analysis

Two aberrant karyotypes were observed, with X chromosome monosomy detected in 4–5 of the 20 metaphase diffuses analyzed. These structural abnormalities were detected in both non-genome-edited TIL samples and TALEN-edited TIL samples, therefore, these abnormalities cannot be attributed to TALEN-mediated genome editing. Furthermore, as assessed in a cytogenetic analysis of 200 metaphase cells, no significant increase in mutagenesis was observed in PD-1 TALEN-edited TILs compared to unmodified TILs. Based on these results, it is concluded that no evidence of TALEN-induced clonal chromosomal aberrations was found in any of the tested samples, and therefore, no significant genotoxicity was identified following TALEN-mediated genome editing.

Example 21: Exemplary TIL Manufacturing Process

Figures 74A-F depict an exemplary TIL manufacturing process. Briefly, on day 0, tumor tissue is isolated from hypothermosol, the bioload sample is stored in transport medium, and tumor fragments are seeded into multiple (2, 3, or 4) G-Rex 100MCS culture flasks at a density ≤50 fragments/flask. Excess fragments are rapidly frozen. In some embodiments, the activation step can be incorporated into the pre-REP step, which provides a better co-stimulatory environment for TILs within the rosette/tumor ME. For example, on day 3, 60 μg of OKT3 or TransAct is added to each of the multiple G-Rex 100MCS culture flasks, and the cells undergo initial activation. On days 7/8, the volume is reduced, the sample is filtered, and transferred to a pooled EXP1000. The sample is removed for cell counting/viability analysis. Cells are washed, centrifuged at 400 g, 20 °C for 10 min, and aliquoted into TALEN and control samples at a ratio ≥9:1. Cells were resuspended in T buffer and electroporated at 10 × ^10⁶ cells/cup with TALEN mRNA (TALEN sample) or without RNA (control sample). Electroporation control and TALEN samples from each cup were seeded into G-Rex100M culture flasks containing 100 mL of medium (containing 6000 IU/mL IL-2) and incubated at 37°C for 1 hour. Feeder cells were irradiated, thawed, collected, and incubated with IL-2. Samples were removed for cell counting and viability analysis. Feeder cells and additional IL-2 and OKT3 were added to the incubated control and TALEN samples in G-Rex 100MCS culture flasks to generate REP cultures (1x G-Rex 100MCS for control REP culture, ≤9x G-Rex 100MCS for TALEN REP culture). On days 10/11, 500 mL of culture medium along with 3000 IU/mL IL-2 was added to each culture flask of the REP culture. Alternatively, the cell culture medium could be completely replaced with fresh medium. This step did not require transferring the cell suspension and did not use G-Rex 500MCS. On day 16, the volume was reduced, and the samples were collected. The samples were transferred via a blood filter, and the samples were removed for cell counting/viability analysis. Control samples were centrifuged, and the control final formulation was prepared and cryopreserved at a controlled rate. TALEN samples were processed using the LOVO system, and the TALEN residue final formulation was prepared and cryopreserved at a controlled rate. The prepared product samples were then subjected to quality control analysis.

This process has the following advantages: a) it eliminates the suspension transfer step for activation in the bag; b) it allows for 1/10 scale control; c) it eliminates in-process transfer to G-Rex10; d) it eliminates the overnight incubation step at 30°C, allowing for immediate reactivation (REP) of the medium at ambient temperature; and e) it eliminates one treatment day.

Example 22: A Phase 1/2 Study Evaluating the Efficacy and Safety of PD-1KO TIL Infusion

introduction

The phase 1/2 open-label study of PD-1 knockout tumor-infiltrating lymphocyte (PD-1KO TIL) products was conducted in participants with unresectable or metastatic melanoma or stage III or IV non-small cell lung cancer.

PD-1KO TIL product is an autologous TIL product that has undergone genome editing with transcription activator-like effector nucleases to disrupt the gene PDCD1 encoding PD-1. This protocol will evaluate PD-1KO TIL product as a treatment option for participants with unresectable or metastatic melanoma or stage III or IV NSCLC.

Research Principles

This study marks the first human investigation of the PD-1KO TIL product. The antitumor activity of the PD-1KO TIL product and its ability to directly target and kill tumor cells in a manner similar to non-genome-edited autologous TIL products were evaluated, with the potential for enhanced antitumor activity due to PDCD1 disruption.

Consistent with the hypothesis, enhanced antitumor activity of the PD-1KO TIL product was observed relative to non-genome-edited autologous TILs in a patient-derived xenograft mouse model. A proof-of-concept nonclinical study of the PD-1KO TIL product further demonstrated that TALEN-mediated PDCD1 mutagenesis was reproducible, correlated with reduced PD-1 cell surface expression, and indicated that the general biological characteristics of autologous TILs considered crucial for antitumor activity were either unaffected or positively affected by PD-1 gene knockout. In the absence of conventional nonclinical animal models associated with human TIL testing, in vitro studies supported the safety of the PD-1KO TIL product. Low off-target activity was detected at one of the top 20 potential off-target sites. Given the low mutation frequency and the fact that this site corresponds to a non-coding RNA not known to be associated with the disease, this is unlikely to pose a safety risk to the product. Additional in vitro studies showed no TALEN-related clonal chromosomal abnormalities following TALEN-mediated genome editing.

background

Unresectable or metastatic melanoma, as well as stage III and IV NSCLC, represent malignancies with a significant unmet medical need. Despite recent advances in melanoma treatment, including ICIs (e.g., anti-cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4] and anti-PD-1 monoclonal antibodies [mAb]) and targeted therapies (e.g., proto-oncogene B-Raf [BRAF] and mitogen-activated protein kinase [MEK] inhibitors), unresectable or metastatic melanoma remains difficult to treat and continues to be a major public health problem (Howlader 2020; Steininger 2021). While several effective NSCLC therapies are currently available, including ICIs (i.e., nivolumab and pembrolizumab), platinum-based double chemotherapy, and tyrosine kinase inhibitors (TKIs) or other targeted therapies against driver mutation-positive tumors, none have provided adequate activity and durable cancer control. Ultimately, most tumors progress despite the initial effectiveness of the available therapies. Therefore, despite the approval of ICI, there remains a significant unmet medical need for melanoma and NSCLC patients who have made progress on available standard care therapies.

Autologous TILs have demonstrated durable responses and have the potential to address a major unmet need in clinical trial participants with metastatic melanoma who have limited treatment options, including those with primary refractory to anti-PD-1/PD-L1 therapy (Rosenberg 2011; Larkin 2021; Sarnaik 2021). Similarly, preliminary data suggest that TILs elicit clinically meaningful responses in NSCLC participants who have experienced disease progression following a series of therapies, including ICIs, and in participants with epidermal growth factor receptor (EGFR) driver mutation-positive tumors (Creelan 2021; Schoenfeld 2021).

The antitumor activity of TILs can be inhibited through interactions between lymphocytes and the tumor microenvironment. Specifically, signaling that occurs when PD-1 on the surface of T cells binds to PD-L1 on tumor cells negatively impacts T cell antitumor activity. Consistent with this finding, various antibody therapies that specifically block the PD-1/PD-L1 pathway, such as the anti-PD-1 antibodies pembrolizumab and nivolumab, have shown potent activity in solid tumors, both as single agents and in combination with non-genome-edited autologous TILs. Notably, in addition to their promising anticancer activity, clinical studies of non-genome-edited autologous TIL therapy combined with anti-PD-1 antibodies in unresectable or metastatic melanoma, recurrent and/or metastatic head and neck squamous cell carcinoma, and persistent, recurrent, or metastatic cervical cancer provide preliminary evidence of the safety of PD-1 signaling inactivation in the context of adoptive cell therapy (ACT) with TILs, suggesting a safety profile consistent with that of individual therapies (O'Malley 2021). This is consistent with the results of other studies conducted to date in participants with metastatic melanoma (evaluating the combination of TIL therapy and immune checkpoint inhibition) (Besser 2013; Mullinax 2018) and a study investigating the administration of TIL therapy in combination with nivolumab in participants with NSCLC (Creelan 2021).

Systemic administration of anti-PD-1 antibodies generates adverse events (AEs) due to the non-specific upregulation of immune pathways via T cell activation and proliferation. As a potential alternative to this treatment, TALEN genome editing technology is used to specifically inactivate PDCD1 in autologous TILs, thereby producing PD-1KO TIL products, PD-1 gene knockout products. By limiting the inhibition of PD-1 signaling in therapeutic tumor-derived T cells, PD-1KO TIL product therapy is expected to provide a safer alternative to ICI therapy, which imparts systemic inhibition of the PD-1 pathway. Furthermore, compared to anti-PD-1 antibody regimens requiring repeated administration, PD-1KO TIL product therapy has the potential to offer clinical benefit as a one-time treatment.

Benefit/Risk Assessment

Currently, no clinical safety data are available for the PD-1KO TIL product, as this is the first human study of the PD-1KO TIL product. There are no clinical safety data from studies of ACT and autologous TILs with PD-1 gene knockout via genome editing; however, safety data are available from non-genome-edited TIL regimens administered alone or in combination with anti-PD-1 antibodies. Safety data from studies of non-genome-edited TIL products are considered relevant because they are manufactured using a derivative process of the PD-1KO TIL product manufacturing process, have the same formulation as the PD-1KO TIL product, and are administered as part of a regimen similar to the proposed PD-1KO TIL product regimen.

The risk and benefit assessment presented below is based on observations related to the administration of NMA-LD and IL-2, as reported in their respective prescribing information, and clinical experience with similar treatments to PD-1KO TIL products, specifically non-genome-edited autologous TIL products administered alone or in combination with anti-PD-1 antibodies, as part of a regimen similar to the proposed PD-1KO TIL product regimen.

Benefit assessment

Potential benefits to participants include the possibility of receiving a research intervention that could stop, slow, or reverse cancer progression in patient populations lacking effective treatment options.

Overall Benefits and Risks Conclusion

Considering the following, the benefit-risk profile of the PD-1KO TIL product treatment regimen is suitable for participants planned to be enrolled in the IOV-GM1-201 study:

●The known toxicities of non-genome-edited autologous TIL regimens (i.e., NMA-LD, IL-2, and non-genome-edited autologous TIL products) administered alone or in combination with anti-PD-1 antibodies are monitorable and manageable.

● Measures taken to minimize the risk to participants

● The lack of effective treatment options that provide durable benefits to the study population, and

●This new therapy has the potential to improve the antitumor activity and safety profile of existing TIL-based solid tumor treatment regimens and PD-1 inhibitor therapies.

Goals and End Points

Research Design

Overall Design

This is a phase 1/2, open-label, non-randomized, multi-cohort, multicenter study with a safety lead-in period, evaluating autologous TIL regimens using the PD-1KO TIL product in participants with unresectable or metastatic melanoma (cohort 1) or stage III or IV NSCLC (cohort 2). In each cohort, tumor samples were excised from each participant and cultured in vitro to produce IOV-4001. Participants were infused with the PD-1KO TIL product, followed by IL-2, after lymphocyte depletion chemotherapy including cyclophosphamide and fludarabine. During the safety lead-in period, the first three participants from either cohort will receive treatment in a staggered manner. Once each participant successfully completes the 28-day DLT observation period (starting at the time of PD-1KO TIL product infusion), the next participant may be authorized to continue with NMA-LD. Subsequent recruitment will follow a standard 3+3 downgrade design, allowing additional participants to join while evaluating the safety and tolerability data of the emerging PD-1KO TIL product. These safety data will be reviewed by the SRC, which comprises representatives of the sponsor and researchers from all recruited DLT-evaluable participants, to advise on dose level decisions. Overall safety will be monitored by an independent DSMB. Recruitment for both cohorts will continue after the first 3 to 6 participants have completed the DLT observation period and received DSMB approval; there will be no staggered periods between participants.

Scientific Principles of Research Design

The study was designed without a control group because these participants, who had undergone extensive pretreatment, had no alternative effective treatment options that could provide durable benefits to the study population. Although no comparisons were available, given the nature of the participants' advanced, refractory disease and the fact that the study intervention (autologous TIL regimen) was administered as a single, one-time therapy, any major tumor regression observed during the study could be presumed to be attributable to the study intervention.

The primary efficacy endpoint of the second phase of this study, namely the ORR according to RECIST v1.1, is the validated method for evaluating tumor response in a single-arm study.

Study population

Prospective approval of deviations from recruitment criteria is not permitted; this is also known as program waiver or exemption.

Key inclusion criteria

Participants are eligible to be included in the study only if they meet all of the following criteria:

1. Participants must be at least 18 years old when signing the informed consent form.

2. Participants who were histologically or pathologically diagnosed with unresectable or metastatic melanoma of stage IIIC, IIID, or IV (cohort 1) or stage III or IV NSCLC (cohort 2).

3. Participants who have previously received the following treatments:

Cohort 1 (Melanoma): Participants who experienced documented radiographic disease progression during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibody. If the tumor was BRAF V600 mutation-positive, participants also received a BRAF inhibitor with or without a MEK inhibitor.

Cohort 2 (NSCLC): Participants who had previously received no more than 3 lines of prior therapy, and

● Participants without oncogene-driven tumors who experienced documented progression of radiation-related disease during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibodies.

● Or for participants with tumors driven by oncogenes for which effective targeted therapies are available (e.g., EGFR, anaplastic lymphoma kinase [ALK], ROS proto-oncogene [ROS], or proto-oncogenes encoding MET tyrosine receptor kinase [MET]), who have experienced documented radiological disease progression during or after at least one line of appropriate targeted therapy and platinum-based dual chemotherapy, or during systemic therapy with PD-1/PD-L1 blocking antibodies, or within 12 weeks after the last dose of a PD-1/PD-L1 blocking antibody.

4. Participants whose ECOG fitness level is 0 or 1.

5. Participants assessed as having at least one resectable lesion

6. Previously irradiated lesions must have radiographic progression before collection.

7. Participants who are of childbearing age or whose partners are of childbearing age must be willing to use an approved, highly effective method of contraception during the treatment period and for 12 months after receiving all protocol-related treatments.

Key Exclusion Criteria

Participants were excluded from the study if they met any of the following criteria:

1. Participants with severe heart disease.

2. Participants with uveal/ocular melanoma.

3. Participants with symptomatic and untreated brain metastases.

4. Participants with any form of primary immunodeficiency.

5. Participants who have had another primary malignant disease within the previous 3 years.

6. Participants who have received or will receive a live or attenuated vaccine within 28 days prior to the start of NMA-LD.

References for Example 22

Besser MJ, Shapira-Frommer R, ltzhaki O, et al. Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma: Intent-to-Treat Analysis and Efficacy after Failure to Prior Immunotherapies. ClinCancer Res. 2013; 19(17): 4792-800.

Creelan BC, Wang C, Teer J, et al. Tumor-Infiltrating LymphocyteTreatment for Anti-PD-1-Resistant Metastatic Lung Cancer: A Phase 1 Trial. NatMed. 2021; 27(8): 1410-1418.

Howlader N, Noone A, Krapcho M, et al. 2020. SEER Cancer Statistics Review, 1975-2017. National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2017/, based on November 2019 SEER data submission.

Larkin JMG, Sarnaik A, Chesney JA, et al. Lifileucel(LN-144), aCryopreserved Autologous Tumor Infiltrating Lymphocyte(TIL)Ther apy inPatients with Advanced Melanoma: Evaluation of Impact of Prior Anti-PD-1Therapy.J Clin Oncol.2021;39(15_suppl):9505.

Mullinax JE, Hall M, Prabhakaran S, et al. Combination of lpilimumab and Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes for Patients withMetastatic Melanoma. Front Oncol. 2018; 8:44.

O'Malley D, Lee SM, Psyrri A, et al.Phase 2Efficacy and Safety ofAutologous Tumor-Infiltrating Lymphocyte(TIL)Cell Therapy in Combinati on with Pembrolizumab in Immune Checkpoint Inhibitor-Patients with Advanced Cancers J Immunother Cancer. 2021; 9(suppl 2, abs492): A523-4.

Rosenberg SA, Yang JC, Sherry RM, et al. Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell TransferImmunotherapy. Clin Cancer Res. 2011;17(13):4550-7.

Sarnaik AA, Hamid O, Khushalani NI, et al. Lifileucel, a Tumor-Infiltrating Lymphocyte Therapy, in Metastatic Melanoma. J Clin Oncol. 2021: JCO2100612.

Schoenfeld AJ, Lee SM, Paz-Ares L, et al. First Phase 2Results of Autologous Tumor-Infiltrating Lymphocyte (TIL; LN-145) Monotherapy in Patient swith Advanced, Immune Checkpoint Inhibitor-Treated, Non-Small Cell Lung Cancer (NsCLC). J Immunother Cancer. 2021; 9 (suppl2, abs 458): A486-7.

Steininger J, Gellrich FF, Schulz A, et al. Systemic Therapy of Metastatic Melanoma: On the Road to 75Cure. Cancers (Basel). 2021; 13(6).

Example 23: Evaluation of Xenon Electroporation Instrument

A study was conducted to determine whether a thermoexcimer could be used in the process of generating PD-1KO TILs. The results are summarized in Figure 76.

Method Overview

Activated or pre-REP TILs are used for experiments.

●REP TIL: Demonstration Day, Neon Experiment 1, Xenon Experiment 1

●Pre-REP TIL: Xenon Experiment 3, Xenon Experiment 4

●Activate for 5 days using TransAct

Cell preparation days for electroporation (same across experiments):

● Equilibrate the buffer solution to room temperature.

● Wash TIL with 1x PBS

● Wash TIL with 1x electroporation buffer (Thermo-CTS ^™ Xenon ^™ Genome Editing Buffer)

● Centrifuge and resuspend in the final volume of electroporation buffer.

○ A single injection requires an exact volume of 1 ml per sample (maximum 100e6/ml).

○ The neon volume can be 10ul or 100ul (maximum 20e6/ml).

●Add mRNA (GFP 1ug/1e6 cells, TALEN 4ug/1e6 cells)

●Electroplution

● Rest overnight in CM2 + 3000 IU/ml IL-2 in G-Rex10 (if using >10e6, split into 2 G-Rex10s).

●37℃, for GFP mRNA

●30℃, for TALEN mRNA

● Collect resting samples and bacterial strains using a 150µm cell filter.

● Perform cell counting and flow staining or REP (Xenon Experiment 4).

Experiment 1: Demonstration Day, Thermo

● Activated REP TIL (L4346) was tested using GFP mRNA.

● Thawing (Day 0) and Activation (Day 1) 50e6+TransAct

● 1ug/1e GFP mRNA per 6 cells (stock solution, 1mg/ml)

● Electroporation using Thermo's recommended optimization scheme

● Single injection (range: 2e7-1e8 cells per 1ml) – 20e6/ml

●Neon – 2e6/100ul

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 77, the results indicate that after 24 hours, 1700/20/1 and 2300/3/4 showed higher GFP expression and recovery rates.

Experiment 2: Neon Experiment 1

●Objective: To optimize two electroporation setups identified during the xenon demonstration using neon and compare them with BTX.

●Cell concentration: 2e6 cells per 100 μl of neon-1

● Xenon backbone electroporation setup:

○1700/20/1

○2300/3/4

● Change the duration (ms) and number of pulses while keeping the voltage setting constant.

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 78, the results indicate that the 2300/3/4 Thermo recommended setting (5*) has the highest GFP expression and 60% recovery rate, and the recovery rate is improved at the 2300V voltage setting with shorter duration (2ms) and fewer pulses (3), and GFP expression is reduced.

Experiment 3: Xenon Experiment 1

●Objective: To determine the effect of cell concentration on xenon-GFP expression and recovery rate.

●N = 1 activated REP TIL sample (EP11231)

●Xenon and BTX

●Electroporation conditions: 2300/3/4, selected based on maximum GFP expression and 60% recovery rate.

●Use a single injection to test cell concentration

○1e6/ml

○5e6/ml

○10e6/ml

○25e6/ml

●BTX Comparison

○10e6+GFP (in 400 μl, therefore the concentration is 25e6/ml)

○2e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 79, the results indicate that GFP expression was slightly reduced when xenon was used compared to BTX. GFP expression was not affected by cell concentration, but the recovery rate decreased and apoptosis increased at lower cell concentrations. After 24 hours, 2300/3/4 appeared to be toxic at low cell concentrations.

Experiment 4: Xenon Experiment 3

Objective: To determine the effect of cell concentration (low/high) on xenon GFP expression and recovery rate using the EP setting that exhibits the highest recovery rate.

●N = 1 activated pre-REP TIL sample (K7136)

●Xenon and BTX

●Electroplution conditions:

○1400/30/1

○1700/20/1

○2300/2/3

○2500/2/5

●Use a single injection to test cell concentration

○5e6/ml

○25e6/ml

●BTX Comparison

○5e6+GFP (in 200 μl, therefore the concentration is 25e6/ml)

○1e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 80, the results indicate that high GFP expression exists between samples, comparable to that of BTX, and the difference between 5e6 and 25e6 is minimal. For EP settings 1400/30/1 and 2300/2/3, there are similar recovery rates between 5e6 and 25e6, and after 24 hours, the recovery rate of 2300/2/3 is comparable to that of BTX.

Experiment 5: Xenon Experiment 4

●Objective: To compare the Xenon electroporator and the BTX electroporator using TALEN mRNA.

●N = 1 activated pre-REP TIL sample (L4374)

●Xenon and BTX

●Electroporation conditions:

○2300/2/3

●Conditions:

Xenon (Talen(25e6))

Xenon simulant (5e6)

○BTX Talen (25e6)

○BTX emulator (5e6)

●Test parameters:

○Resuscitation rate and survival rate

○ PD-1 KO based on flow cytometry

■ PD-1 phase KO flow cytometry data operated on resting samples after electroporation (without REP)

■REP collects final PD-1KO flow cytometry data from samples

As shown in Figure 81, the results showed that the recovery rates of xenon and BTX were similar between 5e6 and 25e6, with xenon having a slightly higher recovery rate. The PD-1 KO efficiency (resting cells after electroporation) was similar between xenon (89%) and BTX (86%). Furthermore, for the analysis of PD-1 expression and KO efficiency in REP samples, the KO efficiency of xenon (83%) was slightly higher than that of BTX (76%).

in conclusion

The study identified an electroporation setting of 2300/2/3 for xenon that resulted in high cell resuscitation rates (comparable to BTX) at both low and high cell concentrations, high GFP expression (comparable to BTX) at both low and high cell concentrations, and high PD-1 KO efficiency (comparable to BTX).

The above embodiments are provided to give those skilled in the art a complete disclosure and description of how to prepare and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications to the above-described patterns of the invention that are obvious to those skilled in the art are intended to fall within the scope of the following claims. All patents and disclosures mentioned in this specification indicate the skill level of those skilled in the art to which this invention pertains.

All headings and section titles are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different headings and sections as needed, in accordance with the spirit and scope of the invention described herein.

All references cited in this article are incorporated herein by reference in their entirety for all purposes, as if each individual publication or patent or patent application were specifically and individually indicated to be incorporated herein by reference in their entirety for all purposes.

As will be apparent to those skilled in the art, various modifications and alterations can be made thereto without departing from the spirit and scope of this application. The specific embodiments and examples described herein are provided by way of example only, and this application is limited only to the appended claims and the full scope of their equivalents.

Methods for generating TIL products using PD-1 TALEN gene knockdown

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 63/242,373, filed September 9, 2021; U.S. Provisional Application No. 63/287,670, filed September 9, 2021; U.S. Provisional Application No. 63/322,190, filed March 21, 2022; U.S. Provisional Application No. 63/354,605, filed June 22, 2022; and U.S. Provisional Application No. 63/394,248, filed August 1, 2022, the disclosures of which are incorporated herein by reference in their entirety.

Background of the Invention

Using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) to treat large, refractory cancers represents an effective approach for treating patients with poor prognoses. (Gattinoni et al., Nat. Rev. Immunol. 2006, 6, 383-393). TILs are predominantly T-cells, and IL-2-based TIL expansion followed by a “rapid expansion process” (REP) is the preferred method for TIL expansion due to its speed and efficiency. (Dudley et al., Science 2002, 298, 850-54; Dudley et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. Immunother. 2003, 26, 332-42.) Numerous approaches have been explored to improve the response of melanoma to TIL therapy and to extend TIL therapy to other tumor types, but success has been limited and the field remains challenging. (Goff et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg et al., Clin. Cancer Res. 2011, 17, 4550-57.) Combination studies with single immune checkpoint inhibitors have also been described, but further investigation is underway and additional treatment options are needed (Kverneland et al., Oncotarget, 2020, 11(22), 2092-2105).

Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, aseptic issues, and other factors described herein, severely restricting their therapeutic potential for patients refractory to checkpoint inhibitor therapy. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes, suitable for treating patients with few or no viable treatment options. This invention addresses this need by providing a shortened manufacturing process for generating TILs.

The present invention provides improved and/or shortened processes and methods for amplifying TILs and generating therapeutic TIL populations, including methods for gene editing at least a portion of the therapeutic TIL population to enhance its therapeutic efficacy.

Summary of the Invention

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing of at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, the culturing or rapid second expansion of the fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the fourth TIL population into multiple subcultures; each subculture being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs or a therapeutic TIL population.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the activation of the second TIL population is performed using anti-CD3 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using OKT-3.

In some implementations, the step of activating the second TIL population is performed using 300 ng/mL of OKT-3.

In some implementations, the step of activating the second TIL population is performed using anti-CD3 agonist and anti-CD28 agonist beads or antibodies.

In some implementations, the step of activating the second TIL population is performed using TransAct.

In some implementations, the step of activating the second TIL population is performed using TransAct diluted at 1:10, 1:17.5, or 1:100.

In some implementations, the step of activating the second TIL population takes about 2 days.

In some implementations, the step of activating the second TIL population takes about 3 days.

In some implementations, the step of activating the second TIL population takes approximately 4 days.

In some implementations, the step of activating the second TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the fourth TIL population takes about 8 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 9 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the step of culturing the fourth TIL population takes about 10 days.

In some implementations, the step of culturing the fourth TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some implementations, the method further includes:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A second TIL population was produced by culturing a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium for about 3–9 days in a first cell culture medium containing IL-2 and OKT-3;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some implementations, the step of culturing or rapidly expanding the third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the third TIL population into multiple subcultures; each of the subcultures being cultured in a third cell culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the step of culturing the first TIL population takes about 3 days.

In some implementations, the step of culturing the first TIL population takes about 5 days.

In some implementations, the step of culturing the first TIL population takes about 7 days.

In some implementations, the step of culturing the third TIL population takes about 8 days.

In some implementations, the step of culturing the third TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes about 8-9 days.

In some implementations, the step of culturing the third TIL population takes about 10 days.

In some implementations, the step of culturing the third TIL population takes about 8-10 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, the step of culturing or initially amplifying the first TIL population in a first culture medium also includes anti-CD3 and anti-CD28 beads or antibodies.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the step of culturing or initially expanding the first TIL population includes culturing the first TIL population for about 3 days in a first cell culture medium containing IL-2 and anti-CD3 and anti-CD28 beads or antibodies, followed by culturing the first TIL population for 2-4 days in a cell culture medium containing IL-2 and OKT-3.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies contain TransAct.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

In some embodiments, the first culture medium contains 300 ng/mL of OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of at least two gene editors is transferred independently of the transfer of any other gene editor during the electroporation step.

In some implementations, the electroporation step also includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing IL-2 and/or IL-15.

In some embodiments, the first and second resting periods comprise incubating a third or fourth TIL population in a second cell culture medium containing 300 IU/mL, 1000 IU/mL, or 6000 IU/mL of IL-2.

In some embodiments, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a second cell culture medium containing 15 ng/mL IL-15.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first and second resting periods comprise incubating a third or fourth TIL population at approximately 25, 28, 30, 32, 35, or 37°C with approximately 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some implementations, the electroporation step is preceded by washing a second or third TIL population in a cell pore buffer.

In some embodiments, the at least one gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates PD-1 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CTLA-4 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates LAG-3 expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CISH expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates CBL-B expression.

In some embodiments, the at least one gene editor comprises a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CTLA-4.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and TIGIT.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and LAG-3.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CISH.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CBL-B.

In some implementations, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CISH and CBL-B.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, the tumor tissue is processed into multiple tumor fragments.

In some implementations, the tumor fragment is added to the closed system.

In some embodiments, 150 or fewer fragments, 100 or fewer fragments, or 50 or fewer fragments are added to the closed system. 167. A gene-edited tumor-infiltrating lymphocyte (TIL) population comprising an expanded TIL population, wherein the expression of at least one protein is regulated by a gene editor transferred to at least a portion of the expanded TIL population.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the at least one protein is PD-1.

In some embodiments, the at least one protein is CTLA-4.

In some embodiments, the at least one protein is LAG-3.

In some embodiments, the at least one protein is CISH.

In some embodiments, the at least one protein is CBL-B.

In some embodiments, the at least one protein is TIGIT.

In some embodiments, the expression of the at least two proteins is regulated by at least two gene editors transferred to at least a portion of the amplified TIL population, wherein the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of the first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of the second protein.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first TALE protein and the second TALE protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some implementations, the gene-edited TIL populations disclosed herein are created using the methods disclosed herein.

In some embodiments, this document provides a pharmaceutical composition comprising the gene-edited TIL population disclosed herein and a pharmaceutically acceptable vector.

In some implementations, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of the gene-edited TIL population disclosed herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) The tumor fragment is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step includes delivering a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CTLA-4 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting PD-1 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and LAG-3 expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CTLA-4 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of the TALEN system for inhibiting CTLA-4 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CISH expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting LAG-3 and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and CBL-B expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CISH and TIGIT expression.

In some embodiments, the electroporation step includes delivery of a TALEN system for inhibiting CBL-B and TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is about 1× ^10⁹ to about 1× ^10¹¹ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of the collected TIL population in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the patient has been given a therapeutically effective dose of the collected TIL population in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Attached Figure Description

Figure 1: Exemplary Gen 2 (Process 2A) diagram, which provides an overview of steps A through F.

Figures 2A to 2C: Process flow diagrams of one implementation of Gen 2 (process 2A) for TIL manufacturing.

Figure 3: A diagram showing one embodiment of an exemplary manufacturing process for cryopreserved TILs (approximately 22 days).

Figure 4: A diagram showing one implementation of Gen 2 (Process 2A, a 22-day process for TIL manufacturing).

Figure 5: Comparison table of steps A to F of an exemplary implementation of process 1C and Gen 2 (process 2A) for TIL manufacturing.

Figure 6: A detailed comparison of one implementation of Process 1C and one implementation of Gen 2 (Process 2A) for TIL manufacturing.

Figure 7: Exemplary Gen 3 type TIL manufacturing process.

Figures 8A through 8P: A) A comparison between an implementation of the 2A process (approximately 22 days) and a Gen 3 process (approximately 14 to 16 days) for TIL manufacturing. B) An exemplary Gen 3 process diagram providing an overview of steps A through F (approximately 14 to 16 days). C) Diagrams of three exemplary Gen 3 processes, outlining steps A through F (approximately 14 to 16 days) for each of the three process variations. D) An exemplary modified Gen 2-like process providing an overview of steps A through F (approximately 22 days). E-P) Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 9: Experimental flowchart providing comparability between Gen 2 (process 2A) and Gen 3 processes.

Figure 10: A comparison of various Gen 2 (Process 2A) and Gen 3.1 process implementation schemes.

Figure 11: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 12: An overview of the culture medium conditions for one implementation of the Gen 3 process (referred to as Gen 3.1).

Figure 13: A table describing the various features of the implementation schemes for the Gen 2, Gen 2.1, and Gen 3.0 processes.

Figure 14: A table comparing various features of the implementation schemes for the Gen 2 and Gen 3.0 processes.

Figure 15: A table showing the culture medium used in various implementation schemes of the described amplification process.

Figure 16: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 17: A schematic diagram of an exemplary implementation of a method for amplifying T cells from hematopoietic malignancies using the Gen 3 amplification platform.

Figure 18: Structures IA and IB are provided. The cylinder refers to an individual polypeptide-binding domain. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from, for example, 4-1BBL or antibodies binding to 4-1BB, which fold to form a trivalent protein. This trivalent protein is then linked to a second trivalent protein via an IgG1-Fc (including CH3 and CH2 domains), which in turn links the two trivalent proteins together via disulfide bonds (small elongated ellipses). This stabilizes the structure and provides an agonist capable of aggregating the intracellular signaling domains of six receptors with signaling proteins to form a signaling complex. The TNFRSF-binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys sequences.

Figure 19: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 20: Process overview of an exemplary implementation of the Gen 3.1 process (16-day process).

Figure 21: A schematic diagram of an exemplary implementation of the Gen 3.1 testing process (16-day to 17-day process).

Figure 22: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 23: Comparison table of exemplary Gen 2 and exemplary Gen 3 processes.

Figure 24: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day to 17-day process) preparation timeline.

Figure 25: A schematic diagram of an exemplary implementation of the Gen 3 process (14-day to 16-day process).

Figures 26A to 26B: Schematic diagrams of an exemplary implementation of the Gen 3 process (16-day process).

Figure 27: A schematic diagram of an exemplary implementation of the Gen 3 process (16-day process).

Figure 28: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 29: Comparison of one implementation scheme of Gen 2, Gen 2.1 and Gen 3 processes (16-day process).

Figure 30: Components of the Gen 3 implementation scheme.

Figure 31: Comparison of Gen 3 implementation flowcharts (Gen 3.0, Gen 3.1 comparison, Gen 3.1 test).

Figure 32: Shows components of an exemplary implementation of the Gen 3 process (16- to 17-day process).

Figure 33: Acceptance Criteria Table.

Figure 34: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figure 35: Experimental flowchart of the full-scale PD-1KO TIL TALEN process.

Figures 36A to 36D: Schematic diagrams of exemplary implementations of the KO TIL TALEN process.

Figure 37: A schematic diagram of an exemplary implementation of the process described in Example 12.

Figures 38A-38B: In vivo efficacy of PDCD-1KO TIL. A) PDCD-1KO efficiency as assessed by flow cytometry. B) hIL-2NOG mice with melanoma tumor cells transplanted via adoptive transfer using PDCD-1KO or TIL-mimicking mice (n=14 per treatment group). Anti-PD-1 antibody treatment combined with TIL-mimicking mice served as a control for PD-1/PD-L1 blockade. Statistical significance is indicated by *p<0.05, **p<0.01, and ****p<0.0001.

Figures 39A to 39E: TIL product analysis. A) Live cell dose, B) Purity, C) Identity, D) Potency, and E) PDCD-1KO efficiency of TIL products.

Figures 40A to 40B: TIL product analysis. A) TIL differentiation and B) TIL memory.

Figures 41A to 41B: Expression of activation and inhibition of related biomarkers on PDCD-1KO TIL.

Figures 42A to 42B: IL-2-independent proliferation assay of PDCD-1KO TIL products.

Figure 43: Summary of karyotype analysis results of PDCD-1KO TIL products.

Figures 44A to 44B: Cell viability (Figure 44A) and recovery fold (Figure 44B) before electroporation.

Figures 45A to 45B: Cell recovery fold (Figure 45A) and cell viability (Figure 45B) after electroporation.

Figures 46A to 46C: Gene knockout efficiency of CD3+ (Figure 46A), CD8+ (Figure 46B), and CD4+ (Figure 46C) cells.

Figures 47A to 47B: Cell recovery fold (Figure 47A) and cell viability (Figure 47B) after electroporation.

Figures 48A to 48B: Cell recovery fold (Figure 48A) and cell viability (Figure 48B) after electroporation when using 6000 IU/mL IL-2.

Figures 49A to 49B: Cell recovery fold (Figure 49A) and cell viability (Figure 49B) after electroporation under various conditions.

Figures 50A to 50C: Gene knockout efficiency of CD3+ (Figure 50A), CD8+ (Figure 50B), and CD4+ (Figure 50C) cells.

Figure 51: Cell viability before electroporation.

Figure 52: Cell resuscitation fold before electroporation.

Figures 53A to 53B: Cell recovery fold (Figure 53A) and cell viability (Figure 53B) after electroporation.

Figures 54A to 54C: Gene knockout efficiency of CD3+ (Figure 54A), CD8+ (Figure 54B), and CD4+ (Figure 54C) cells.

Figures 55A to 55B: Cell number (Figure 55A) and survival rate (Figure 55B) after various washing steps.

Figures 56A to 56B: Cell numbers after various rotation conditions using PBS washing (Figure 56A) or Cyto washing (Figure 56B).

Figures 57A to 57B: Cell viability after various rotation conditions using PBS washing (Figure 57A) or Cyto washing (Figure 57B).

Figures 58A to 58B: Total rotation comparison of cell number (Figure 58A) and total rotation comparison of cell survival rate (Figure 58B) after various rotation conditions.

Figure 59: Percentage of cell loss after total rotation under various rotation conditions.

Figures 60A to 60C: Percentage of cell loss and survival rate during electroporation, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation.

Figures 61A to 61C: Gene knockout efficiency of CD3+ (Figure 61A), CD8+ (Figure 61B), and CD4+ (Figure 61C) cells.

Figures 62A to 62B: Cell viability (Figure 62A) and fold expansion (Figure 62B) of the REP collection.

Figures 63A to 63B: Percentage of cell loss after electroporation (Figure 63A) and cell viability (Figure 63B).

Figures 64A to 64C: Gene knockout efficiency of CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells.

Figures 65A to 65B: Folding of REP collection (Figure 65A) and cell viability (Figure 65B).

Figures 66A to 66C: Cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C).

Figure 67: Percentage of growth after 3 days of rest.

Figures 68A to 68C: PD-1 gene knockout efficiency.

Figure 69: PDCD1 gene modification according to NGS.

Figures 70A to 70B: Distribution of the TCR Vβ isotype in the CD3+PD-1- subset in the main PD-1KO TIL product and NE TIL.

Figures 71A to 71B: PD-1KO TIL effect function as measured by MLR (Figure 71A) and multifunctionality (Figure 71B).

Figure 72: In vivo antitumor activity of M1152 PD-1KO TIL product.

Figures 73A to 73B: Durability of TALEN protein in autologous TILs as a function of time, measured by Western blot.

Figures 74A-F: Exemplary TIL manufacturing process.

Figures 75A-B: Schematic diagrams of the 1/2 phase study described in Example 22.

Figure 76: Summary of data described in Example 23.

Figures 77A-D: Results of the demonstration day experiment from Example 23.

Figures 78A-C: Results of neon experiment 1 from Example 23.

Figures 79A-C: Results of Xenon Experiment 1 from Example 23.

Figures 80A-B: Results of Xenon Experiment 3 from Example 23.

Figures 81A-C: Results of Xenon Experiment 4 from Example 23.

A brief explanation of sequence listings

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of moromumab.

SEQ ID NO:3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO:4 is the amino acid sequence of adefovir.

SEQ ID NO:5 is in IL-2 form.

SEQ ID NO:6 is the amino acid sequence of Nevo interleukin-α.

SEQ ID NO:7 is in IL-2 form.

SEQ ID NO:8 is a mucin domain polypeptide.

SEQ ID NO:9 is the amino acid sequence of recombinant human IL-4 protein.

SEQ ID NO:10 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO:11 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO:12 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO:13 is an IL-2 sequence.

SEQ ID NO:14 is the sequence of the IL-2 mutant protein.

SEQ ID NO:15 is the sequence of the IL-2 mutant protein.

SEQ ID NO:16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

SEQ ID NO:17 is HCDR2 of IgG.IL2R67A.H1.

SEQ ID NO:18 is HCDR3 of IgG.IL2R67A.H1.

SEQ ID NO:19 is HCDR1_IL-2kabat of IgG.IL2R67A.H1.

SEQ ID NO:20 is HCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:21 is HCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:22 is HCDR1_IL-2clothia of IgG.IL2R67A.H1.

SEQ ID NO:23 is HCDR2 clothia of IgG.IL2R67A.H1.

SEQ ID NO:24 is HCDR3 clothia of IgG.IL2R67A.H1.

SEQ ID NO:25 is HCDR1_IL-2IMGT of IgG.IL2R67A.H1.

SEQ ID NO:26 is HCDR2 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:27 is HCDR3 IMGT of IgG.IL2R67A.H1.

SEQ ID NO:28 is the V _H chain of IgG.IL2R67A.H1.

SEQ ID NO:29 is the heavy chain of IgG.IL2R67A.H1.

SEQ ID NO:30 is the LCDR1 kabat of IgG.IL2R67A.H1.

SEQ ID NO:31 is the LCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO:32 is the LCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO:33 is the LCDR1 chothia of IgG.IL2R67A.H1.

SEQ ID NO:34 is the LCDR2 chothia of IgG.IL2R67A.H1.

SEQ ID NO:35 is the LCDR3 chothia of IgG.IL2R67A.H1.

SEQ ID NO:36 is the V _L chain.

SEQ ID NO:37 is a light chain.

SEQ ID NO:38 is a light chain.

SEQ ID NO:39 is a light chain.

SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

SEQ ID NO:41 is the amino acid sequence of mouse 4-1BB.

SEQ ID NO:42 is the heavy chain of utomilumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:43 is the light chain of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:44 is the heavy chain variable region (V _H ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:45 is the light chain variable region (V _L ) of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:46 is the heavy chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:47 is the heavy chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:48 is the heavy chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:49 is the light chain CDR1 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:50 is the light chain CDR2 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:51 is the light chain CDR3 of utumumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:52 is the heavy chain of urelumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:53 is the light chain of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:54 is the heavy chain variable region (VH) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:55 is the light chain variable region (VL) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:56 is the heavy chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:57 is the heavy chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:58 is the heavy chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:59 is the light chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:60 is the light chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:61 is the light chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO:62 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:63 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:64 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:65 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:66 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:67 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:68 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:69 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:70 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:71 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:72 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:73 represents the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO:74 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:75 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:76 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO:77 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

SEQ ID NO:78 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO:79 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 1.

SEQ ID NO:80 is the light chain variable region (V _L ) of type 1 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:81 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 of type 2.

SEQ ID NO:82 is the light chain variable region (V _L ) of type 2 of 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO:83 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:84 is the light chain variable region (V _L ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO:85 is the amino acid sequence of human OX40.

SEQ ID NO:86 is the amino acid sequence of mouse OX40.

SEQ ID NO:87 is the heavy chain of the OX40 agonist monoclonal antibody taboliziumab (MEDI-0562).

SEQ ID NO:88 is the light chain of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:89 is the heavy chain variable region (V _H ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:90 is the light chain variable region (V _L ) of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:91 is the heavy chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:92 is the heavy chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:93 is the heavy chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:94 is the light chain CDR1 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:95 is the light chain CDR2 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:96 is the light chain CDR3 of tavoricumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO:97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:99 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:100 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO:107 is the heavy chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:108 is the light chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO:109 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:110 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO:117 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:118 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:119 is the heavy chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:120 is the heavy chain CDR2 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:122 is the light chain CDR1 of Hu119-122, an OX40 agonist monoclonal antibody.

SEQ ID NO:123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO:125 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:126 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:130 is the light chain CDR1 of Hu106-222, an OX40 agonist monoclonal antibody.

SEQ ID NO:131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO:133 is the amino acid sequence of the OX40 ligand (OX40L).

SEQ ID NO:134 is the soluble portion of the OX40L polypeptide.

SEQ ID NO:135 is an alternative soluble fraction of the OX40L polypeptide.

SEQ ID NO:136 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:137 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO:138 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:139 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO:140 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 021.

SEQ ID NO:141 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 021.

SEQ ID NO:142 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:143 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 023.

SEQ ID NO:144 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:145 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:146 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:147 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:148 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:149 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:150 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:151 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:152 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:153 is the heavy chain variable region ( _VH ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:154 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:155 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO:156 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:157 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:160 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:161 is the amino acid sequence of the light chain variable region ( _VL ) of the PD-1 inhibitor nivolumab.

SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:170 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:171 is the amino acid sequence of the light chain variable region (V _L ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:180 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:181 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:190 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:191 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor avirumab.

SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avirumab.

SEQ ID NO:198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:200 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:201 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:210 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:211 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:220 is the amino acid sequence of the heavy chain variable region (V _H ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:221 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor trimexumab.

SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor trimemumab.

SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4 inhibitor zeflimab.

SEQ ID NO:230 is the amino acid sequence of the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:231 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zefirimab.

SEQ ID NO:238 is an exemplary Clo05 l nuclease domain amino acid sequence.

SEQ ID NO:239 is an exemplary piggyBac(PB) transposase amino acid sequence.

SEQ ID NO:240 is an exemplary Sleeping Beauty transposase amino acid sequence.

SEQ ID NO:241 is an exemplary amino acid sequence of an overactive Sleeping Beauty (SB100X) transposase.

I. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and disclosures mentioned herein are incorporated herein by reference in their entirety.

As used herein, the terms "co-administration/co-administering," "administered in combination with/administering in combination with," "simultaneous," and "concurrent" encompass the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, such as multiple TILs) to a subject, such that both the active pharmaceutical ingredient and/or its metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of separate compositions and administration of a composition in which two agents are present are preferred.

The term "in vivo" refers to events that occur within the body of a subject.

The term "in vitro" refers to an event that occurs outside the subject's body. In vitro assays encompass cell-based assays using live or dead cells, and may also include cell-free assays that do not use intact cells.

The term "ex vivo" refers to an event involving the treatment or procedure performed on cells, tissues, and/or organs that have been removed from a subject's body. Appropriately, cells, tissues, and/or organs may be returned to the subject using surgical or therapeutic methods.

The term "rapid amplification" means that the number of antigen-specific TILs increases at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) within one week, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) within one week, or most preferably at least about 100-fold within one week. Several rapid amplification protocols are described herein.

In this article, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are TILs obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any expanded or proliferated population of TIL cells as discussed herein, including (but not limited to) main TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations may contain genetically modified TILs.

In this article, "cell population" (including TILs) refers to a large number of cells sharing common characteristics. Typically, the population size ranges from 1 × ^10⁶ to 1 × ^10¹⁰ cells, with different TIL populations containing varying numbers. For example, initial growth of primary TILs in the presence of IL-2 produces a main TIL population of approximately 1 × ^10⁸ cells. REP expansion is typically performed to provide a cell population of 1.5 × ^10⁹ to 1.5 × ^10¹⁰ cells for infusion.

In this document, “cryopreserved TIL” means TIL that has been processed and stored in the range of approximately -150°C to -60°C, whether primary, host, or amplified (REP TIL). General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, “cryopreserved TIL” may be distinguished from frozen tissue samples that can be used as a source of primary TIL.

In this article, “thawed cryopreserved TILs” means a population of TILs that were previously cryopreserved and then processed to be restored to room temperature or higher (including, but not limited to, cell culture temperatures or temperatures at which TILs can be administered to patients).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to reintroduce into patients with late-stage invasive solid tumors.

The term "cryopreservation media" refers to any culture medium that can be used for cryopreservation of cells. Such media may include those containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 media may be referred to by the trade name "CS10". CS10 media is a serum-free, animal-free medium containing DMSO. In some embodiments, CS10 media contains 10% DMSO.

The term "central memory T cells" refers to a subset of T cells in humans that are CD45R0+ and constitutively express CCR7 (CCR7 ^hi ) and CD62L (CD62 ^hi ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Upon TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are mainly found in the CD4 compartments of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

The term "effective memory T cells" refers to a subset of human or mammalian T cells, such as central memory T cells, which are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 ^lo ) and are heterogeneous or low in expression of CD62L (CD62L ^lo ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, upon antigen stimulation. Effector memory T cells are primarily found in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry a large amount of perforin.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, for example (but not limited to), closed G containers. Once a tumor segment is added to the closed system, the system remains closed to the external environment until the TIL is ready for administration to the patient.

As used herein, the terms “fragmenting,” “fragment,” and “fragmented” describe the process of destroying a tumor, including mechanical fragmentation methods such as crushing, slicing, splitting, and pulverizing tumor tissue, as well as any other methods used to destroy the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBL is separated from whole blood or apheresis products from a donor. In some embodiments, PBL is separated from whole blood or apheresis products from a donor by positively or negatively selecting for a T cell phenotype (e.g., CD3+CD45+ T cell phenotype).

The term "anti-CD3 antibody" refers to an antibody or a variant thereof that targets the CD3 receptor in the T-cell antigen receptor of mature T cells, such as a monoclonal antibody, and includes human, humanized, chimeric, murine, or mammalian antibodies. Anti-CD3 antibodies include OKT-3, also known as moromuzumab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof targeting the CD3 receptor in the T-cell antigen receptor of mature T cells, including human, humanized, chimeric, or murine antibodies, and including commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, San Diego, CA, USA) and moromumab or its variants, conserved amino acid substitutions, glycosylated forms, or biosimilars. The amino acid sequences of the heavy and light chains of moromumab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). Hybridomas capable of producing OKT-3 are deposited at the American Type Culture Collection and designated ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited at the European Collection of Authenticated Cell Cultures (ECACC) with catalog number 86022706.

Table 1. Amino acid sequence of moromuzumab (exemplary OKT-3 antibody)

The term "IL-2" (also referred to herein as "IL2") refers to a T-cell growth factor called interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in this invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses recombinant human IL-2, such as adefovir (PROLEUKIN, available from multiple suppliers, 22 million IU per single-use vial), as well as recombinant IL-2 forms supplied by CellGenix (CELLGRO GMP) of Portsmouth, NH, USA, or ProSpec-Tany TechnoGene Ltd. (catalog number CYT-209-b) of East Brunswick, NJ, USA, and other commercial equivalents from other suppliers. Adefovir (desalanyl-1, serine-125 human IL-2) is a non-glycosylated recombinant human IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of the adefovir suitable for use in this invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including the PEGylated IL-2 prodrug beempegaldesleukin (NKTR-214, as SEQ ID NO:4, PEGylated recombinant human IL-2 wherein an average of 6 lysine residues are substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluorene-9-yl)methoxy]carbonyl N ⁶ ), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO 2018/132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US2019/0275133 A1, the disclosure of which is incorporated herein by reference. The bepedelion interleukin (NKTR-214) and other PEGylated IL-2 molecules applicable to the present invention are described in U.S. Patent Application Publication No. US2014/0328791A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 applicable to the present invention are described in U.S. Patents Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations applicable to the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

In some embodiments, the IL-2 form suitable for use in this invention is THOR-707, available from Synthorx. The preparation and characteristics of THOR-707 and additional alternative forms of IL-2 suitable for use in this invention are described in U.S. Patent Application Publications Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for use in this invention is an interleukin-2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugate portion bound to the isolated and purified IL-2 polypeptide at amino acid positions selected from the following: K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the amino acid residues are numbered corresponding to SEQ ID NO: 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residues are mutated to cysteine. In some embodiments, the amino acid residues are mutated to lysine. In some embodiments, amino acid residues selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are further mutated to non-natural amino acids. In some embodiments, the non-natural amino acids include N6-azidoethoxy-L-lysine (AzK), N6-propynylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, 3-methyl-phenylalanine, L-DOPA, fluorinated phenylalanine, Isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzoxy)-3-oxopropyl)amino)ethyl)selenoyl)propionic acid, 2-amino-3-(phenylselenoyl)propionic acid, or selenocysteine. In some embodiments, the IL-2 conjugate exhibits reduced affinity for the IL-2 receptor α (IL-2Rα) subunit relative to the wild-type IL-2 peptide. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% of the binding affinity to IL-2Rα. In some embodiments, the reduced affinity relative to the wild-type IL-2 peptide is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 100, 200, 300, 500, 1000, or greater. In some embodiments, the conjugated moiety weakens or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugation portion comprises a water-soluble polymer. In some embodiments, each water-soluble polymer independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each water-soluble polymer independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises polydextrose, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each water-soluble polymer independently comprises a polysaccharide. In some embodiments, the water-soluble polymers each independently comprise a polyamine. In some embodiments, the conjugated moiety comprises a protein. In some embodiments, an additional conjugated moiety comprises a protein. In some embodiments, the proteins each independently comprise albumin, transferrin, or transthyretin. In some embodiments, the proteins each independently comprise an Fc moiety. In some embodiments, the proteins each independently comprise the Fc moiety of IgG. In some embodiments, the conjugated moiety comprises a peptide. In some embodiments, an additional conjugated moiety comprises a peptide. In some embodiments, the peptides each independently comprise XTEN peptides, high-amino acid polymers rich in glycine (HAP), PAS peptides, elastin-like peptides (ELP), CTP peptides, or gelatin-like protein (GLK) polymers. In some embodiments, the isolated and purified IL-2 peptide is modified by glutamylation. In some embodiments, the conjugated moiety binds directly to the isolated and purified IL-2 peptide. In some embodiments, the conjugated moiety binds indirectly to the isolated and purified IL-2 peptide via a linker. In some embodiments, the linker comprises a homologous bifunctional linker. In some embodiments, the homodifunctional linker comprises Lomant's reagent, dithiobis(succinimide propionate) DSP, 3'3'-dithiobis(succinimide propionate sulfonyl succinimide) (DTSSP), succinimide disuccinate (DSS), succinimide disuccinate (BS), succinimide tartrate (DST), disulfonyl tartrate (sulfonyl DST), glycosylbis(succinimide succinate) ethylene ester (EGS), glutarate succinimide (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl hexamethylenediimide (DMA), dimethyl heptamethine (DMP), dimethyl octanediimide (DMS), dimethyl-3,3'-dithiobispropionide (DSP) DTBP), 1,4-bis(3'-(2'-pyridyldithio)propamido)butane (DPDPB), bis(cis-butenyldiimide)hexane (BMH), compounds containing aryl halides (DFDNB) (e.g., 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the connector comprises a heterogeneous bifunctional connector. In some embodiments, the heterogeneous bifunctional connector comprises 3-(2-pyridyldithio)propionic acid N-succinimide ester (sPDP), long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (LC-sPDP), water-soluble long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (sulfonyl-LC-sPDP), succinimide-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), or sulfosuccinimide-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate. (Sulfon-LC-sMPT), succinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimide-4-(N-cis-butenyldiimidemethyl)cyclohexane-1-carboxylate (sulfon-sMCC), m-cis-butenyldiimide benzoyl-N-hydroxysuccinimide (MBs), m-cis-butenyldiimide benzoyl-N-hydroxysulfosuccinimide (sulfon-MBs), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB), (4-iodoacetyl)aminobenzoic acid N-succinimide (sIAB) ) sulfosuccinimide aminobenzoate (sulfon-sIAB), succinimide-4-(p-cis-butenylimide-phenyl)butyrate (sMPB), sulfosuccinimide-4-(p-cis-butenylimide-phenyl)butyrate (sulfon-sMPB), N-(γ-cis-butenylimide-butyryloxy)succinimide (GMBs), N-(γ-cis-butenylimide-butyryloxy)sulfosuccinimide (sulfon-GMBs), 6-((iodoacetyl)amino)hexanoic acid succinimide (sIAX), 6-[6-(((iodoacetyl)amino)hexanoic acid succinimide Acetyl)amino)hexanoyl)amino]hexanoic acid succinimide ester (sIAXX), 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylic acid succinimide ester (sIAC), 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoic acid succinimide ester (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl reactive and thiohydrogen-reactive crosslinking agents, such as 4-(4-N-cis-butenyldiimide-phenyl)butyric acid hydrazide (MPBH), 4-(N-cis-butenyldiimide-methyl)cyclohexane- 1-Carboxy-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfon-NHs-AsA), sulfosuccinimide-(4-azidosalicylamido)hexanoate (sulfon-NHs-LC-AsA), sulfosuccinimide-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimide-4 -Azide benzoate (HsAB), N-hydroxysulfosuccinimide-4-azidobenzoate (sulfon-HsAB), N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfon-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimide-2-(m-azido-o-nitrobenzoamide)-ethyl-1,3' -Dithiopropionate (sAND), N-succinimide-4-(4-azidophenyl)1,3'-dithiopropionate (sADP), (4-azidophenyl)-1,3'-dithiopropionate N-sulfosuccinimide (sulfon-sADP), 4-(p-azidophenyl)butyrate sulfosuccinimide (sulfon-sAPB), 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate sulfosuccinimide (sAED), 7-azido-4-methylcoumarin-3-acetic acid sulfosuccinimide (sulfon-s... AMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleic anhydride group, optionally comprising a maleic anhydride hexanoyl (mc), succinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimide-4-(N-maleicimide methyl)cyclohexane-1-carboxylate (sulfon-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives thereof, or the like. In some embodiments, the conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, an additional conjugation moiety is capable of prolonging the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in this invention is polyethylene glycol-modified as disclosed in U.S. Patent Application Publication US 2020/0181220 A1 and U.S. Patent Application Publication US 2020/0330601 A1. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion relative to one residue of SEQ ID NO:5. In some embodiments, the IL-2 form suitable for the present invention lacks the IL-2Rα linker but maintains normal binding to the intermediate affinity IL-2Rβ-γ signaling complex. In some embodiments, the IL-2 form suitable for the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5; and AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in this invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:5; and an AzK substitute for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, referring to the amino acid positions in SEQ ID NO:5.

In some embodiments, the IL-2 form suitable for use in this invention is Nevor interleukin-α (also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes). Nevo interleukin-α, also known as the human interleukin-2 fragment (1-59) variant (Cys ¹²⁵ > Ser ⁵¹ ), is fused to the human interleukin-2 fragment (62-132) via a peptide linker ( ⁶⁰ GG ⁶¹ ), which is fused to the human interleukin-2 receptor α chain fragment (139-303) via a peptide linker ( ¹³³ GSGGGS ¹³⁸ ). It is produced in Chinese hamster ovary (CHO) cells and is glycosylated. The human interleukin-2 (IL-2) (75-133)-peptide [Cys ¹²⁵ (51) > Ser]-mutant (1-59) is fused to the human interleukin-2 (IL-2) (4-74)-peptide (62-132) via a _G2 peptide linker (60-61) and via GSG _3. The S-peptide linker (133-138) is fused to the human interleukin 2 receptor α chain (IL2R subunit α, IL2Rα, IL2RA) (1-165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, in glycoform α. The amino acid sequence of Nevo interleukin α is provided in SEQ ID NO:6. In some embodiments, Nevo interleukin α exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbers in SEQ ID NO:6), and glycosylation sites at the following positions: N187, N206, T212 (using the numbers in SEQ ID NO:6). The preparation and properties of Nevo interleukin-α, as well as additional alternative forms of IL-2 suitable for the present invention, are described in U.S. Patent Application Publication No. US2021/0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the IL-2 form suitable for the present invention has the amino acid sequence provided in SEQ ID NO:6 or its conserved amino acid substitutions. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, the IL-2 form suitable for the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner via a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity with IL-1Rα and having receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises an immunoglobulin comprising all or part of an Fc region, wherein the mucin domain polypeptide linker comprises an amino acid sequence of SEQ ID NO:8 or having at least 90% sequence identity with SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

Table 2. Amino acid sequence of interleukin.

In some embodiments, the IL-2 form suitable for use in this invention comprises an antibody cytokine transplantation protein comprising: a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells relative to regulatory T cells. In some implementations, the IL-2 regimen comprises administering the antibody described in U.S. Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine transplantation protein comprises: a heavy chain variable region (VH) including complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region (VL) including LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or fragment thereof transplanted into a CDR of the _VH or _VL , wherein the IL-2 molecule is a mutant protein, wherein the antibody cytokine transplantation protein preferentially amplifies T effector cells relative to regulatory T cells, and wherein the antibody further comprises an IgG heavy chain and an IgG light chain selected from the group consisting of: an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:38; an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:29; an IgG light chain comprising SEQ ID NO:39 and an IgG heavy chain comprising SEQ ID NO:29; and an IgG light chain comprising SEQ ID NO:37 and an IgG heavy chain comprising SEQ ID NO:38.

In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR1 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR2 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into HCDR3 of _VH , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR1 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR2 of _VL , wherein the IL-2 molecule is a mutant protein. In some embodiments, an IL-2 molecule or a fragment thereof is transplanted into LCDR3 of _VL , wherein the IL-2 molecule is a mutant protein.

The insertion of an IL-2 molecule can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine transplantation protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence replaces all or part of the CDR sequence. The IL-2 molecule replacement can occur at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. The IL-2 molecule replacement can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

In some embodiments, the IL-2 molecule is directly grafted into a CDR without a peptide linker, wherein there are no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted into a CDR with a peptide linker, wherein there are one or more additional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecule described herein is an IL-2 mutant protein. In some cases, the IL-2 mutant protein contains an R67A substitution. In some embodiments, the IL-2 mutant protein contains the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutant protein contains the amino acid sequence of Table 1 in U.S. Patent Application Publication No. US 2020/0270334A1, the disclosure of which is incorporated herein by reference.

In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16. In some embodiments, the antibody cytokine transplantation protein comprises HCDR1 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine transplantation protein comprises HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine transplantation protein comprises a _VH region containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine transplantation protein includes a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a light chain containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a _VH region containing the amino acid sequence of SEQ ID NO:28, and a _VL region containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:29, and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO:38, and a light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain region containing the amino acid sequence of SEQ ID NO:38; and a light chain region containing the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplantation protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 or variants, derivatives, or fragments thereof, or conserved amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with them, as described in U.S. Patent Application Publication No. 2020/0270334A1. In some embodiments, the antibody component of the antibody cytokine transplantation protein described herein comprises the immunoglobulin sequence, framework sequence, or CDR sequence of palizumab. In some embodiments, the serum half-life of the antibody cytokine transplantation protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to aldehyde interleukin or similar molecules). In some embodiments, the antibody cytokine transplantation protein described herein has a sequence as illustrated in Table 3.

Table 3: Sequence of exemplary palizumab antibody-IL-2 transplanted protein

The term “IL-4” (also referred to herein as “IL4”) refers to a cytokine known as interleukin-4, produced by Th2 T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and _IgG1 expression. Recombinant human IL-4 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-211) and ThermoFisher Scientific of Waltham, MA, USA (recombinant human IL-15 protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for this invention is provided in Table 2 (SEQ ID NO:9).

The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine called interleukin-7, which is available from stromal and epithelial cells as well as dendritic cells. (Fry and Mackall, Blood 2002, 99, 3892-904). IL-7 stimulates T cell development. IL-7 binds to the IL-7 receptor (a heterodimer composed of IL-7 receptor α and a common γ chain receptor), which belongs to a family of signaling pathways important for T cell development within the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in this invention is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-254) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for this invention is provided in Table 2 (SEQ ID NO: 10).

The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor called interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the contents of which are incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-230-b) and ThermoFisher Scientific of Waltham, Massachusetts (recombinant human IL-15 protein, catalog number 34-8159-82). The amino acid sequence of the recombinant human IL-15 suitable for use in this invention is provided in Table 2 (SEQ ID NO: 11).

The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycosylated forms, biosimilars, and their variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the contents of which are incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, New Jersey (catalog number CYT-408-b) and Thermo Fisher Scientific of Waltham, Massachusetts (recombinant human IL-21 protein, catalog number 14-8219-80). The amino acid sequence of the recombinant human IL-21 suitable for use in this invention is provided in Table 2 (SEQ ID NO:21).

When indicated as “effective antitumor dose,” “effective tumor-suppressing dose,” or “therapeutic dose,” the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease. Generally, it can be stated that the pharmaceutical composition described herein, comprising tumor-infiltrating lymphocytes (e.g., passaged TILs or genetically modified cytotoxic lymphocytes), can be administered at doses of ^10⁴ to ^10¹¹ cells/kg body weight (e.g., ^10⁵ to ^10⁶ , ^10⁵ to ^10¹⁰ , ^10⁵ to ^10¹¹ , ^10⁶ to ^10¹⁰ , ^{10⁶ to 10¹¹ , 10⁷} to ^10¹¹ , ^10⁷ to ^10¹⁰ , 10⁸ to ^10¹¹ , ^10⁸ to ^10¹⁰ , ^10⁹ to ^10¹¹ , or ^10⁹ to ^10¹⁰ cells/kg body weight), including all integer values within the range. TIL (in some cases, including genetically modified cytotoxic lymphocytes) compositions may also be administered at these doses multiple times. TILs (including genetically engineered TILs in some cases) can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). The optimal dosage and treatment regimen for a particular patient can be readily determined by medical professionals by monitoring the patient’s disease symptoms and adjusting the treatment accordingly.

The term "hematological malignancy" or related terms refer to cancers and tumors of the hematopoietic and lymphoid tissues of mammals (including, but not limited to, tissues of the blood, bone marrow, lymph nodes, and lymphatic system). Hematological malignancies are also known as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancy" refers to hematological malignancies affecting B cells.

The term "liquid tumor" refers to an abnormal clump of cells that is fluid in nature. Liquid tumor cancers include (but are not limited to) leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs derived from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs derived from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

As used herein, the term “microenvironment” can refer to the solid or hematologic tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanotransmitters that “promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, encourage treatment resistance, and provide a niche for the vigorous growth of dominant metastases,” as described by Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression of the microenvironment.

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (on days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m²/day for 5 days (on days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours to achieve physiological tolerance.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for components of the immune system (“cytokine pool”) prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as “immunosuppressive modulation”) in a patient prior to the introduction of the TIL of the present invention.

The term "effective amount" or "therapeutic effective amount" refers to an amount of compound or combination of compounds as described herein that is sufficient to achieve the intended application, including (but not limited to) the treatment of a disease. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo) or the subject being treated and the disease condition (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. The term also applies to doses that will induce a specific response in target cells (e.g., reduced platelet adhesion and/or cell migration). Specific doses will vary depending on: the specific compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

The terms “treatment”, “treating”, “treat”, etc., refer to achieving a desired pharmacological and/or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of partial or complete cure of a disease and/or adverse effects attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development or progression; and (c) alleviating the disease, i.e., causing disease remission and/or alleviating one or more disease symptoms. “Treatment” is also intended to cover the delivery of agents to provide a pharmacological effect, even in the absence of a disease or ailment. For example, “treatment” covers the delivery of compositions that can induce or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

The terms “non-myeloablative chemotherapy,” “non-myeloablative lymphocyte depletion,” “NMALD,” “NMA LD,” “NMA-LD,” and any variations thereof are used interchangeably to indicate a chemotherapy regimen designed to deplete a patient’s lymphoid immune cells while avoiding depletion of their myeloid immune cells. Typically, a patient receives one cycle of non-myeloablative chemotherapy before receiving tumor-infiltrating lymphocytes as described herein.

When used in conjunction with parts of a nucleic acid or protein, the term "heterologous" indicates that the nucleic acid or protein contains two or more subsequences that are not found in nature to be identical to each other. For example, nucleic acids are often produced in a recombinant manner, having two or more sequences from unrelated genes arranged to create new functional nucleic acid sequences, such as a promoter from one source and a coding region from another source or coding regions from different sources. Similarly, a heterologous protein indicates a protein that contains two or more subsequences that are not found in nature to be identical to each other (e.g., fusion proteins).

In the context of two or more nucleic acids or polypeptides, the terms "sequence identity," "percent identity," and "sequence percent identity" (or their synonyms, such as "99% identical") refer to two or more sequences or subsequences that are identical or have the same specific percentage of nucleotide or amino acid residues when compared and aligned (introducing gaps where necessary) to achieve maximum correspondence and without considering any conserved amino acid substitutions as part of the sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. Suitable programs for determining the percentage of sequence identity include, for example, the BLAST package available from the U.S. Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California, USA), or MegAlign (available from DNASTAR) are other publicly available software programs that can be used for sequence alignment. Those skilled in the art can determine the appropriate parameters for optimal alignment using specific alignment software. In some embodiments, the default parameters of the alignment software are used.

As used herein, the term "variant" covers (but is not limited to) antibodies or fusion proteins that contain an amino acid sequence different from that of a reference antibody, differing in that one or more substitutions, deletions, and/or additions are present within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conserved substitutions in its amino acid sequence compared to that of the reference antibody. Conserved substitutions may involve, for example, substitutions of similar charged or uncharged amino acids. Variants retain the ability to bind specifically to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

In this document, “tumor-infiltrating lymphocytes” or “TIL” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated into the tumor. TILs include (but are not limited to) CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and subcultured TILs. “Primary TILs” are cells obtained from patient tissue samples as outlined herein (sometimes referred to as “fresh collection”), and “subcultured TILs” are any population of expanded or proliferated TIL cells as discussed herein, including (but not limited to) the main TILs and expanded TILs (“REP TILs”) as discussed herein, as well as “reREP TILs”. reREP TILs may include, for example, a second expanded TIL or a second additional expanded TIL (e.g., the TILs described in step D of Figure 8, including TILs referred to as reREP TILs).

TILs are generally defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and achieve therapeutic effects). TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to be reintroduced into patients after infiltrating solid tumors. TILs can be further characterized by potency; for example, if interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, then TILs can be considered potent. TILs can be considered potent if, for example, interferon (IFNγ) releases greater than approximately 50 pg/mL, greater than approximately 100 pg/mL, greater than approximately 150 pg/mL, or greater than approximately 200 pg/mL, greater than approximately 300 pg/mL, greater than approximately 400 pg/mL, greater than approximately 500 pg/mL, greater than approximately 600 pg/mL, greater than approximately 700 pg/mL, greater than approximately 800 pg/mL, greater than approximately 900 pg/mL, or greater than approximately 1000 pg/mL.

The term "deoxyribonucleic acid" encompasses both natural and synthetic, unmodified and modified deoxyribonucleic acids. Modifications include alterations to the linkages between deoxyribonucleic acids in the sugar moiety, base moiety, and/or oligonucleotides.

The term "RNA" is defined as a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" is defined as a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, RNA produced in a recombinant manner, and altered RNA that differs from naturally occurring RNA through the addition, deletion, substitution, and/or modification of one or more nucleotides. The nucleotides in the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, as well as inert components. The use of such pharmaceutically acceptable carriers or excipients for the active pharmaceutical ingredient is well known in the art. Unless any conventionally pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is covered. Additional active pharmaceutical ingredients, such as those of other pharmaceuticals, may also be incorporated into the described compositions and methods.

The terms “about” and “approximately” mean a range of values that are statistically significant. This range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The permissible differences covered by the terms “about” or “approximately” depend on the specific system under study and are readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not precise and do not need to be precise, but may be approximate and/or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. Generally, whether so explicitly stated or not, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are “about” or “approximately”. It should be noted that embodiments with very different sizes, shapes, and dimensions may employ the described arrangement.

When used in the appended claims in both original and modified forms, the transitional terms “comprising,” “consisting essentially of,” and “consisting of” define the scope of the invention with respect to which additional, unrecited elements or steps (if present) are excluded from the scope of the claims. The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case excludes impurities generally associated with the specified materials. The term “consisting essentially of” limits the scope of the invention to the specified elements, steps, or materials and those elements, steps, or materials that do not substantially affect the basis and novel features of the claimed invention. In alternative embodiments, all compositions, methods, and kits for practicing the invention described herein may be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

The term “antibody” and its plural form “antibodies” refer to a complete immunoglobulin and any antigen-binding fragment (“antigen-binding moiety”) or its single chain. “Antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or its antigen-binding moiety. Each heavy chain includes a heavy chain variable region (abbreviated as _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated as _VL ) and a light chain constant region. The light chain constant region comprises one domain, _CL . The _VH and _VL regions of an antibody can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), and these may be interspersed with more conserved regions called frame regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more epitopes. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen" refers to a substance that induces an immune response. In some embodiments, if presented via a major histocompatibility complex (MHC) molecule, the antigen is a molecule capable of being bound by an antibody or TCR. As used herein, the term "antigen" also encompasses T-cell epitopes. Antigens are additionally capable of being recognized by the immune system. In some embodiments, the antigen is capable of inducing a humoral or cellular immune response that results in the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require the antigen to contain or be linked to a Th-cell epitope. The antigen may also have one or more epitopes (e.g., B epitopes and T epitopes). In some embodiments, the antigen is preferably to react generally with its corresponding antibody or TCR in a highly specific and selective manner, and not with a variety of other antibodies or TCRs that can be induced by other antigens.

The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to formulations of antibody molecules that are single molecular compositions. Monoclonal antibody compositions exhibit single binding specificity and affinity against a specific epitope. Monoclonal antibodies specific to certain receptors can be prepared using knowledge and techniques in the following methods: injecting a suitable antigen into a test subject and then isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. DNA encoding monoclonal antibodies can be isolated and sequenced using routine procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not normally produce immunoglobulins (e.g., *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to obtain monoclonal antibody synthesis in recombinant host cells. The recombinant production of antibodies will be described in more detail below.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Antigen-binding function of antibodies has been demonstrated to be performed by fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of _VL , _VH , _CL , and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; (iii) Fd fragments consisting of _VH and CH1 domains; (iv) Fv fragments consisting of _VL and _VH domains of a single arm of an antibody; (v) domain antibody (dAb) fragments (Ward et al., Nature, 1989, 341, 544-546), which may consist of a _VH or a _VL domain; and (vi) segregated complementarity-determining regions (CDRs). Furthermore, although the two domains ( _VL and _VH ) of the Fv fragment are encoded by independent genes, they can be joined using recombination methods via a synthetic adapter that enables the creation of a single protein chain (referred to as a single-chain Fv (scFv)) in which the _VL and _VH regions pair to form a monovalent molecule; see, for example, Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are targeted at the utility screening fragment in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises both the _VH and _VL portions. A scFv molecule is represented as _VL - _LVH when the _VL domain is the N-terminal portion of the scFv molecule, or as _VH - _LVL when the _VH domain is the N-terminal portion of the scFv molecule. Methods for preparing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB, Vol. 9: 73-80 (1995); and REBird and BWWalker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9: 132-137 (1991), the disclosures of which are incorporated herein by reference.

As used herein, the term "human antibody" is intended to include antibodies that have variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, said constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human framework sequence.

The term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity with a variable region, wherein both the framework and CDR regions of the variable region are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is generated from a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with immortalized cells, having a genome containing both human heavy-chain and light-chain transgenes.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas prepared therefrom (described further below) that are transgenic for the human immunoglobulin gene; (b) antibodies isolated from host cells transformed to express human antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may undergo in vitro mutation induction (or in vivo somatic mutation induction when using transgenic animals with human Ig sequences), and therefore the amino acid sequences of the _VH and _VL regions of the recombinant antibody, although derived from and associated with human germline _VH and _VL sequences, may not be naturally present in the human antibody germline in vivo.

As used in this article, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

The term "human antibody derivative" refers to any modified form of a human antibody, including conjugates of antibodies with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which may be conjugated to the antibody described herein using methods available in the art.

The terms "humanized antibody" and "humanized" refer to antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence. Further frame region modifications can be made to the human frame sequence. Humanized forms of non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. To a large extent, humanized antibodies are human immunoglobulins (receptor antibodies) where residues from the hypervariable region of the receptor are replaced by residues from 15 hypervariable regions of a non-human species (donor antibody) possessing the desired specificity, affinity, and ability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in receptor or donor antibodies. These modifications are performed to further optimize antibody performance. Generally, humanized antibodies will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially all FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein may also be modified to use any Fc variant known to provide effector function and/or FcR binding improvements (e.g., reductions). Fc variants may include any of the amino acid substitutions disclosed below: International Patent Application Publications No. WO 1988/07089A1, WO 1996/14339A1, WO 1998/05787A1, WO 1998/23289A1, WO 1999/51642A1, WO 99/58572A1, WO 2000/09560A2, WO 2000/32767A1, WO 20 No. 00/42072A2, No. WO 2002/44215A2, No. WO 2002/060919A2, No. WO 2003/074569 A2, No. WO 2004/016750 A2, No. WO 2004/029207 A2, No. WO 2004/035752 A2, No. WO 2004/063351A2, No. WO 2004/074455 A2, No. WO 2004/09924 9 A2, WO 2005/040217 A2, WO 2005/070963 A1, WO 2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 A1, WO 2006/047350 A2 and WO 2006/085967 A2; and U.S. Patent No. 5,648,260; Nos. 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the contents of which are incorporated herein by reference.

The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

A “bifunctional antibody” is a small antibody fragment having two antigen-binding sites. The fragment contains a heavy chain variable domain (V_{_H} ), which is linked to a light chain variable domain (V_L ) in the same polypeptide chain (V_{_H}-V_{_L} or V_{_L}-V_{_H}). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, thus creating two antigen-binding sites. Bifunctional antibodies are described in more detail, for example, in European Patent No. 404,097, International Patent Publication No. WO 93/11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

The term "glycosylation" refers to a modified derivative of an antibody. Deglycosylated antibodies are not glycosylated. Glycosylation can be altered to, for example, increase the antibody's affinity for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at said sites. Deglycosylation can increase the antibody's affinity for an antigen, as described in U.S. Patents 5,714,350 and 6,350,861. Alternatively or additionally, antibodies with altered glycosylation types can be produced, such as low-fucosylated antibodies with reduced fucosylation residues or antibodies with increased fractional GlcNac structure. Such altered glycosylation patterns have been shown to enhance antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention to produce glycosylated-modified antibodies. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)fucosyltransferase), so that antibodies expressed in Ms704, Ms705, and Ms709 cell lines do not have fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/- cell lines were formed by breaking the FUT8 gene in targeted CHO/DG44 cells using two alternative vectors (see, for example, U.S. Patent Publication No. 2004/0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation by reducing or eliminating α1,6 bond-related enzymes. The patent also describes cell lines with low or no enzymatic activity for adding fucose to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). International Patent Publication WO 03/035835 describes a variant CHO cell line, namely Lec 13 cells, which has a reduced ability to link fucose to Asn(297)-linked carbohydrates and also induces hypofucosylation of antibodies expressed in said host cells (see also Shields et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication WO 99/54342 describes cells engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine). Cell lines containing GnTIII transferase III (GnTIII) can be used to enhance the antibody's ADCC activity by causing antibodies expressed in engineered cell lines to exhibit an increased bipartite GlcNac structure (see also Umana et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidases can be used to cleave the fucosylate residues of the antibody. For example, α-L-fucosidase removes fucosylate residues from the antibody, as described in Tarentino et al., Biochem. 1975, 14, 5516-5523.

“Polyglycolization” refers to the modification of an antibody or fragment thereof, typically with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that link one or more PEG groups to the antibody or antibody fragment. For example, PEGylation can increase the biological (e.g., serum) half-life of an antibody. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to cover any form of PEG that has been used to derive other proteins, such as mono( _C1 - _C10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-cis-butenediamide. The antibody to be PEGylated is a deglycosylated antibody. Polyethylene glycolation methods are known in the art and applicable to antibodies used in this invention, such as those described, for example, in European Patents EP0154316 and EP0401384 and U.S. Patent No. 5,824,778, the disclosures of which are each incorporated herein by reference.

The term "biosimilar" refers to a biological product (including monoclonal antibodies or proteins) that meets the following criteria: it is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, and there are no clinically significant differences between the biological product and the reference product in terms of safety, purity, and efficacy. Furthermore, a biosimilar or "biosimilar" drug is a biological drug similar to another biological drug authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. A biological product or biological drug is a drug derived from or produced from a biological source (e.g., bacteria or yeast). It may consist of relatively small molecules (e.g., human insulin or erythropoietin) or complex molecules (e.g., monoclonal antibodies). For example, if the reference IL-2 protein is proleukin, then the protein of the reference proleukin approved by a drug regulatory agency is "biosimilar to proleukin" or "a biosimilar of proleukin." In Europe, a biosimilar or “biosimilar” medicine is a biological medicine similar to another biological medicine that has been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for the use of biosimilars in Europe is Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended, and therefore in Europe, biosimilars may be authorized, approved for authorization, or submitted as the subject of authorization pursuant to Article 6 of Regulation (EC) 726/2004 and Article 10(4) of Directive 2001/83/EC. The original biological medicine product authorized in Europe may be referred to as a “reference medicine.” The CHMP guidance on biosimilar products outlines some of the requirements for a product to be considered a biosimilar. In addition, product-specific guidance, including guidance relating to monoclonal antibody biosimilars, is provided by the EMA on a product-by-product basis and published on its website. Biosimilars as described herein may be similar to a reference medicine in terms of quality characteristics, biological activity, mechanism of action, safety profile, and/or efficacy. Furthermore, biosimilars may be used or intended for the treatment of the same disease as the reference medicine. Therefore, biosimilars as described herein can be considered to have similar or very similar quality characteristics to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar biological activity to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar safety profile to the reference drug. Or, or additionally, biosimilars as described herein can be considered to have similar or very similar efficacy to the reference drug. As described herein, in Europe, biosimilars have been compared with reference drugs authorized by the EMA. However, in some cases, in certain studies, biosimilars may be compared with biological products authorized outside the European Economic Area (non-EEA authorized "comparatives"). Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to biological products that have been or may be compared with non-EEA authorized comparatives. Some biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding moieties), and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications (including, for example, amino acid deletions, additions, and/or substitutions) in their amino acid structure that do not significantly affect peptide function. Biosimilars may contain an amino acid sequence having 97% or higher, such as 97%, 98%, 99%, or 100%, sequence identity with the amino acid sequence of a reference drug. Biosimilars may contain one or more post-translational modifications that differ from those of the reference drug, such as (but not limited to) glycosylation, oxidation, deamidation, and/or truncation, provided that such differences do not alter the safety and/or efficacy of the drug. Biosimilars may have the same or different glycosylation patterns as the reference drug. In particular, but not exclusively, biosimilars may have different glycosylation patterns if such differences resolve or are intended to resolve safety concerns associated with the reference drug. Additionally, biosimilars may differ from the reference drug in, for example, their potency, pharmaceutical form, formulation, excipients, and/or delivery method, provided that this does not impair the safety and efficacy of the drug. Compared to a reference product, biosimilars may contain differences in, for example, pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles, but are still considered sufficiently similar to the reference product and thus eligible for or suitable for authorization. In some cases, biosimilars exhibit different binding characteristics compared to the reference product, where the regulatory agency (e.g., the EMA) does not consider these different binding characteristics as an obstacle to authorization of the biosimilar product. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions.

II. Gene Editing Process

A. Overview: TIL amplification + gene editing

Embodiments of the present invention relate to methods for expanding a TIL population, said methods comprising one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, “gene-editing” and “genome editing” refer to a gene modification in which DNA is permanently modified in the genome of a cell, such as by inserting, deleting, modifying, or replacing DNA within the cell's genome. In some embodiments, gene editing causes silencing (sometimes referred to as gene knockout) or suppression/reduction (sometimes referred to as gene knockdown) of DNA sequence expression. According to embodiments of the present invention, gene editing technology is used to enhance the effectiveness of a therapeutic TIL population.

Methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the methods further include gene editing of at least a portion of the TILs. According to other embodiments, methods for expanding TILs into a therapeutic TIL population can be carried out according to any embodiment of the methods described in WO 2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, which are incorporated herein by reference in their entirety, wherein the methods further include gene editing of at least a portion of the TILs. Therefore, one embodiment of the invention provides a therapeutic TIL population expanded according to any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, for example, at least a portion of the therapeutic TIL population transferred to an infusion bag has been permanently gene-edited.

Gene editing during B.TIL amplification

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the method includes the step of culturing or initially expanding a first TIL population, comprising culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days, followed by culturing in a cell culture medium containing IL-2 and OKT-3 for 2-6 days.

In some embodiments, the method includes the steps of culturing or rapidly expanding the third TIL population by culturing the third TIL population in a second cell culture medium for a first period of about 1-7 days, splitting the culture into multiple subcultures at the end of the first period, culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 3-7 days, and merging the multiple subcultures at the end of the second period to provide an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the activation of the second TIL population takes approximately 7 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during, or before, any of steps (a)-(e), (a)-(f), or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e), (a)-(f), or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A third population of TILs was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some embodiments, the step of culturing the third TIL population takes approximately 7 days. In some embodiments, the step of culturing the third TIL population takes approximately 8 days. In some embodiments, the step of culturing the third TIL population takes approximately 9 days. In some embodiments, the step of culturing the third TIL population takes approximately 10 days. In some embodiments, the step of culturing the third TIL population takes approximately 11 days. In some embodiments, the step of culturing the third TIL population takes approximately 12 days. In some embodiments, the step of culturing the third TIL population takes approximately 13 days. In some embodiments, the step of culturing the third TIL population takes approximately 14 days. In some embodiments, the step of culturing the third TIL population takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a third TIL population is performed by culturing the third TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(d) or (a)-(e) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(d) or (a)-(e), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some implementation schemes, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementation schemes, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are then placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to generate a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the first TIL population takes approximately 3-9 days. In some implementations, the step of culturing the first TIL population takes approximately 3-9 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the step of culturing the first TIL population takes approximately 3 days. In some implementations, the step of culturing the first TIL population takes approximately 4 days. In some implementations, the step of culturing the first TIL population takes approximately 5 days. In some implementations, the step of culturing the first TIL population takes approximately 6 days. In some implementations, the step of culturing the first TIL population takes approximately 7 days. In some implementations, the step of culturing the first TIL population takes approximately 8 days. In some implementations, the step of culturing the first TIL population takes approximately 9 days.

In some implementations, the step of culturing the third TIL population takes approximately 1-7 days. In some implementations, the step of culturing the third TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the third TIL population takes approximately 1 day. In some implementations, the step of culturing the third TIL population takes approximately 2 days. In some implementations, the step of culturing the third TIL population takes approximately 3 days. In some implementations, the step of culturing the third TIL population takes approximately 4 days. In some implementations, the step of culturing the third TIL population takes approximately 5 days. In some implementations, the step of culturing the third TIL population takes approximately 6 days. In some implementations, the process of culturing a third TIL population takes about 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed over a period of approximately 22 days. In some embodiments, the steps of the method are completed over a period of approximately 23 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the process of culturing the fourth TIL population takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error in the original.) -13 days, approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 7 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 8 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 9 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 10 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 11 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 12 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 13 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 14 days. In some embodiments, the step of culturing the fourth TIL population takes approximately 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some implementations, the steps of the method are completed over a period of approximately 22 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of the multiple subcultures is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some implementations, the step of culturing the second TIL population takes approximately 2-4 days. In some implementations, the step of culturing the third TIL population takes approximately 2-4 days, approximately 3-4 days, or approximately 2-3 days. In some implementations, the step of culturing the second TIL population takes approximately 2 days. In some implementations, the step of culturing the second TIL population takes approximately 3 days. In some implementations, the step of culturing the second TIL population takes approximately 4 days.

In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 1 day. In some implementations, the step of culturing the fourth TIL population takes approximately 2 days. In some implementations, the step of culturing the fourth TIL population takes approximately 3 days. In some implementations, the step of culturing the fourth TIL population takes approximately 4 days. In some implementations, the step of culturing the fourth TIL population takes approximately 5 days. In some implementations, the step of culturing the fourth TIL population takes approximately 6 days. In some implementations, the step of culturing the fourth TIL population takes approximately 7 days.

In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 3 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 4 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 5 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 6 days. In some embodiments, the step of culturing each of the multiple subculture media is performed for approximately 7 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 3 to 9 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Activate the second TIL population for 1–7 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A rapid second amplification of the fourth TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, methods for preparing expanded tumor-infiltrating lymphocytes (TILs) include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population is performed in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is performed over a period of approximately 1 to 9 days.

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) A rapid second amplification of the third TIL population is performed in a second cell culture medium containing IL-2, OKT-3, and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the rapid second amplification.

In some implementations, initial amplification takes approximately 3-9 days. In other implementations, initial amplification takes approximately 1-9 days, 2-9 days, 3-9 days, approximately 4-9 days, approximately 5-9 days, approximately 6-9 days, approximately 7-9 days, approximately 8-9 days, approximately 1-8 days, approximately 2-8 days, approximately 3-8 days, approximately 4-8 days, approximately 5-8 days, approximately 6-8 days, approximately 7-8 days, approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, initial amplification takes approximately 1 day. In some embodiments, initial amplification takes approximately 2 days. In some embodiments, initial amplification takes approximately 3 days. In some embodiments, initial amplification takes approximately 4 days. In some embodiments, initial amplification takes approximately 5 days. In some embodiments, initial amplification takes approximately 6 days. In some embodiments, initial amplification takes approximately 7 days. In some embodiments, initial amplification takes approximately 8 days. In some embodiments, initial amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the rapid second amplification takes approximately 5-15 days. (The repetition of the repetitions in the original text is likely due to a formatting error or incomplete sentence.) The durations of rapid secondary amplification are approximately 11-13 days, 12-13 days, 5-12 days, 6-12 days, 7-12 days, 8-12 days, 9-12 days, 10-12 days, 11-12 days, 5-11 days, 6-11 days, 7-11 days, 8-11 days, 9-11 days, 10-11 days, 5-10 days, 6-10 days, 7-10 days, 8-10 days, 9-10 days, 5-9 days, 6-9 days, 7-9 days, 8-9 days, 5-8 days, 6-8 days, 7-8 days, 5-7 days, 6-7 days, and 5-6 days. In some implementations, rapid secondary amplification takes approximately 5 days. In some implementations, rapid secondary amplification takes approximately 6 days. In some implementations, rapid secondary amplification takes approximately 7 days. In some embodiments, rapid second amplification takes approximately 8 days. In some embodiments, rapid second amplification takes approximately 9 days. In some embodiments, rapid second amplification takes approximately 10 days. In some embodiments, rapid second amplification takes approximately 11 days. In some embodiments, rapid second amplification takes approximately 12 days. In some embodiments, rapid second amplification takes approximately 13 days. In some embodiments, rapid second amplification takes approximately 14 days. In some embodiments, rapid second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, rapid second amplification is performed by culturing a third TIL population in a second medium for about 5 days in a first period, at which point the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(e) or (a)-(f) outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

According to some embodiments, gene editing is performed while TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some embodiments, gene editing is performed on TILs collected from the culture medium, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, methods for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population include:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digest the tumor tissue sample or tumor fragment in an enzyme medium to produce tumor digest;

(c) The tumor tissue is added to a closed system and a first expansion is performed by culturing a first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using CD3 and CD28 beads or antibodies to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain a third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some implementations, the first amplification takes approximately 3-9 days. In some implementations, the first amplification takes approximately 3-9 days, approximately 3-8 days, approximately 3-7 days, approximately 3-6 days, approximately 3-5 days, approximately 3-4 days, approximately 4-9 days, approximately 4-8 days, approximately 5-9 days, approximately 5-8 days, approximately 6-9 days, approximately 6-8 days, approximately 7-9 days, approximately 7-8 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 3-5 days, approximately 4-5 days, or approximately 3-4 days. In some implementations, the first amplification takes approximately 3 days. In some implementations, the first amplification takes approximately 4 days. In some implementations, the first amplification takes approximately 5 days. In some implementations, the first amplification takes approximately 6 days. In some implementations, the first amplification takes approximately 7 days. In some implementations, the first amplification takes approximately 8 days. In some implementations, the first amplification takes approximately 9 days.

In some implementations, the activation of the second TIL population takes approximately 1-7 days. In some implementations, the activation of the second TIL population takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation of the second TIL population takes approximately 1 day. In some implementations, the activation of the second TIL population takes approximately 2 days. In some implementations, the activation of the second TIL population takes approximately 3 days. In some implementations, the activation of the second TIL population takes approximately 4 days. In some implementations, the activation of the second TIL population takes approximately 5 days. In some implementations, the activation of the second TIL population takes approximately 6 days. In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes approximately 5-15 days. In some implementations, the second amplification takes approximately 5-15 days, approximately 6-15 days, approximately 7-15 days, approximately 8-15 days, approximately 9-15 days, approximately 10-15 days, approximately 11-15 days, approximately 12-15 days, approximately 13-15 days, approximately 14-15 days, approximately 5-14 days, approximately 6-14 days, approximately 7-14 days, approximately 8-14 days, approximately 9-14 days, approximately 10-14 days, approximately 11-14 days, approximately 12-14 days, approximately 13-14 days, approximately 5-13 days, approximately 6-13 days, approximately 7-13 days, approximately 8-13 days, approximately 9-13 days, approximately 10-13 days. The durations of amplification are as follows: approximately 11-13 days, approximately 12-13 days, approximately 5-12 days, approximately 6-12 days, approximately 7-12 days, approximately 8-12 days, approximately 9-12 days, approximately 10-12 days, approximately 11-12 days, approximately 5-11 days, 6-11 days, 7-11 days, approximately 8-11 days, approximately 9-11 days, approximately 10-11 days, approximately 5-10 days, 6-10 days, 7-10 days, approximately 8-10 days, approximately 9-10 days, approximately 5-9 days, 6-9 days, 7-9 days, approximately 8-9 days, approximately 5-8 days, approximately 6-8 days, 7-8 days, approximately 5-7 days, approximately 6-7 days, approximately 5-6 days. In some implementations, the second amplification takes approximately 5 days. In some implementations, the second amplification takes approximately 6 days. In some implementations, the second amplification takes approximately 7 days. In some implementations, the second amplification takes approximately 8 days. In some embodiments, the second amplification takes approximately 9 days. In some embodiments, the second amplification takes approximately 10 days. In some embodiments, the second amplification takes approximately 11 days. In some embodiments, the second amplification takes approximately 12 days. In some embodiments, the second amplification takes approximately 13 days. In some embodiments, the second amplification takes approximately 14 days. In some embodiments, the second amplification takes approximately 15 days.

In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 8 days. In some embodiments, the steps of the method are completed within a period of approximately 9 days. In some embodiments, the steps of the method are completed within a period of approximately 10 days. In some embodiments, the steps of the method are completed within a period of approximately 11 days. In some embodiments, the steps of the method are completed within a period of approximately 12 days. In some embodiments, the steps of the method are completed within a period of approximately 13 days. In some embodiments, the steps of the method are completed within a period of approximately 14 days. In some embodiments, the steps of the method are completed within a period of approximately 15 days. In some embodiments, the steps of the method are completed within a period of approximately 16 days. In some embodiments, the steps of the method are completed within a period of approximately 17 days. In some embodiments, the steps of the method are completed within a period of approximately 18 days. In some embodiments, the steps of the method are completed within a period of approximately 19 days. In some embodiments, the steps of the method are completed within a period of approximately 20 days. In some embodiments, the steps of the method are completed within a period of approximately 21 days. In some embodiments, the steps of the method are completed within a period of approximately 22 days. In some embodiments, the steps of the method are completed within a period of approximately 23 days. In some embodiments, the steps of the method are completed within a period of approximately 24 days. In some embodiments, the steps of the method are completed within a period of approximately 25 days. In some embodiments, the steps of the method are completed within a period of approximately 26 days. In some embodiments, the steps of the method are completed within a period of approximately 27 days. In some embodiments, the steps of the method are completed within a period of approximately 28 days. In some embodiments, the steps of the method are completed within a period of approximately 29 days. In some embodiments, the steps of the method are completed within a period of approximately 30 days. In some embodiments, the steps of the method are completed within a period of approximately 31 days. In some embodiments, the steps of the method are completed within a period of approximately 32 days.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about 5 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the second amplification is carried out by culturing a fourth TIL population in a second medium for about 5 days in a first period, at the end of the first period the culture is split into multiple subcultures, each of which is cultured in a third medium containing IL-2 for about 4 days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about three days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about four days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about five days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about six days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for about one day in a first period, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for about seven days in a second period, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 2 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 3 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 4 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 5 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 6 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 3 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 5 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 6 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some implementations, the step of culturing a fourth TIL population is performed by culturing the fourth TIL population in a second medium for a first period of about 7 days, at the end of the first period the culture is split into multiple subcultures, each of the multiple subcultures is cultured in a third medium containing IL-2 for a second period of about 7 days, and at the end of the second period the multiple subcultures are combined to provide an amplified number of TILs.

In some embodiments, the gene editing process can be performed at any time during the TIL amplification method, meaning that the TILs can be gene-edited before, during, or after any step of the amplification method; for example, during or after any of steps (a)-(f) or (a)-(g) as outlined in the above method. In some embodiments, the gene editing process can be performed more than once at any time during the TIL amplification method. According to some embodiments, TILs are collected during the culture step (e.g., the culture step is “paused” for at least a portion of the TILs), and the gene editing process is performed on the collected TILs, and in some cases, they are subsequently reintroduced into the culture step (e.g., reintroduced into the culture medium) to continue the culture step, such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene-edited.

It should be noted that alternative implementations of the amplification process may differ from the method described above; for example, alternative implementations may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific implementation, the gene editing process can be performed at any time during the TIL amplification method. For example, alternative implementations may include more than two culture steps, and gene editing of the TIL may be performed during the third or fourth culture step, etc.

In some implementations, gene editing is performed while the TILs are still in the culture medium and during the culturing process; that is, they are not necessarily "removed" from the culturing process for gene editing. According to some implementations, TILs collected from the culture medium are gene-edited, and after the gene editing process, the TILs are subsequently placed back into the culture medium.

In some implementations, the step of gene editing at least a portion of the second or third TIL population includes a sterile electroporation step on the second or third TIL population.

In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some embodiments, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step further includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 22 hours. According to some implementations, the quiescent step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the antigen-presenting cells (APCs) are PBMCs. According to some embodiments, the PBMCs are irradiated. According to some embodiments, the PBMCs are allogeneic. According to some embodiments, the PBMCs are both irradiated and allogeneic. According to some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some implementations, the tumor tissue is derived from an autopsied tumor.

In some implementations, the dissected tumor is less than 8 hours old.

In some embodiments, the tumor tissue is selected from the group consisting of: melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple-negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and HPV-positive tumor tissue.

In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 5.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 4.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 3.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 2.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments with a diameter of about 1.5 mm to 2 mm.

In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 1.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 2.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 3.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 4.5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5 mm and a longest side length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular segments with a shortest side length of at least 5.5 mm and a longest side length of about 6 mm.

In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm or about 6 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 3.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 4.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 5.5 mm. In some embodiments, the tumor tissue is fragmented into approximately cubic fragments with sides of about 6 mm.

In some embodiments, the present invention provides a therapeutic population of tumor-infiltrating lymphocyte (TIL) products generated by methods as described herein.

In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising administering to the patient an effective amount of a therapeutic TIL population generated by the method described herein. In some embodiments, the cancer is selected from the group consisting of: glioblastoma (GBM), gastrointestinal cancer, melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, and mantle cell lymphoma. In some implementations, the cancer is selected from the group consisting of: cutaneous melanoma, ocular melanoma, uveal melanoma, conjunctival malignant melanoma, metastatic melanoma, pleomorphic xanthoastrocytoma, dysembryonic neuroepithelial tumor, ganglioglioma and pilocytic astrocytoma, endometrioid adenocarcinoma with significant myxoid differentiation (ECMD), papillary thyroid carcinoma, serous low-grade or borderline ovarian cancer, hairy cell leukemia and Langerhans cell histiocytosis.

In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5000 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 5500 IU/mL and 6000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4000 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 4500 IU/mL and 5000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3000 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 3500 IU/mL and 4000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2000 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 2500 IU/mL and 3000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1000 IU/mL and 2000 IU/mL. In some embodiments, the IL-2 is present in the cell culture medium during the first amplification at an initial concentration between 1500 IU/mL and 2000 IU/mL.

In some embodiments, in the second amplification step, the IL-2 is present at an initial concentration between 1000 IU/mL and 6000 IU/mL, and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some embodiments, the first amplification is performed using a ventilated container. In some embodiments, the second amplification is performed using a ventilated container.

In some embodiments, the first cell culture medium further comprises cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and/or the third culture medium further comprise cytokines selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the method further includes the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated the day after administration of the TIL or PBL product. In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen initiated on the same day as administration of the TIL or PBL product. In some embodiments, the IL-2 regimen comprises adelleukin, nevoleukin, or a bioanalyte or variant thereof.

In some embodiments, the therapeutically effective amount of TIL product comprises about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs.

In some implementations, the number of the second TIL population is at least 50 times that of the first TIL population.

C.PD-1

One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 superfamily of T cell regulators. Its ligands, PD-L1 and PD-L2, are expressed on various tumor cells, including melanoma. The interaction between PD-1 and PD-L1 can suppress T cell effector function, cause T cell exhaustion under chronic stimulation, and induce T cell apoptosis in the tumor microenvironment. PD-1 may also play a role in tumor-specific evasion of immune surveillance.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of PD-1 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the method includes gene editing of at least a portion of the TILs by silencing or inhibiting the expression of PD-1. As described in more detail below, the gene editing process may involve the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., PD-1). For example, the TALEN method can be used to silence or reduce the expression of PD-1 in TILs.

D.CTLA-4

CTLA-4 expression is induced upon T cell activation on activated T cells and competitively binds to antigen-presenting cell activation antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 can induce T cell suppression and is used to maintain the homeostasis of the immune response. However, inhibiting the interaction of CTLA-4 with CD80 or CD86 prolongs T cell activation and thus increases the level of the immune response against cancer antigens.

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of CTLA-4 in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and CTLA-4 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CTLA-4. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CTLA-4). For example, the CRISPR method, the TALE method, or the zinc finger method can be used to silence or inhibit the expression of CTLA-4 in TILs. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and CTLA-4 in TILs.

E.LAG-3

Following conjugation at the major histocompatibility complex (MHC) class II, lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells. Although the mechanism is not fully understood, regulation of this gene can induce negative regulation of T cell function, preventing tissue damage and autoimmunity. Therefore, LAG-3 blockade may improve antitumor responses. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of LAG-3 in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and LAG-3 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of LAG-3. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., LAG-3). According to specific embodiments, the expression of LAG-3 in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and LAG-3 in TILs can be silenced or reduced using the TALEN method.

F.Cish

Cish, a member of the cytokine signaling inhibitory factor (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits its functional affinity for tumors. Deletion of the Cish gene in CD8+ T cells enhances its amplification, functional affinity, and cytokine pluripotency, leading to significant and durable regression of existing tumors. See, for example, Palmer et al., Journal of Experimental Medicine, 212(12):2095 (2015).

According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of Cish in TILs. According to specific embodiments, the compositions and methods according to the invention silence or reduce the expression of both PD-1 and Cish in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of Cish. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., Cish). For example, the expression of Cish in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, the expression of PD-1 and Cish in TILs can be silenced or reduced using the TALEN method.

G.CBL-B

CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and a negative regulator of T cell activation. (Bachmaier et al., Nature, 2000, 403, 211-216; Wallner et al., Clin. Dev. Immunol. 2012, 692-639.)

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of CBL-B in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and CBL-B in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., process 2A or the method shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of CBL-B. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., CBL-B). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, CBL-B is silenced using TALEN gene knockout. In some embodiments, CBL-B is silenced using a TALE-KRAB transcription inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and CBL-B in TIL.

H.TIGIT

TIGIT is a cell surface protein expressed on regulatory, memory, and activated T cells. TIGIT belongs to the poliovirus receptor (PVR) family of immunoglobulins and inhibits T cell activation. (Yu et al., Nat Immunol., 2009, 10(1):48-57).

According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of TIGIT in TILs. According to specific embodiments, the compositions and methods of the present invention silence or reduce the expression of both PD-1 and TIGIT in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the methods shown in Figures 20 and 21), wherein the method includes gene editing of at least a portion of the TIL by silencing or inhibiting the expression of TIGIT. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at immune checkpoint genes (e.g., TIGIT). For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR, TALE, or zinc finger methods. In some embodiments, TIGIT can be silenced using TALEN gene knockout. In some embodiments, TIGIT can be silenced using a TALE-KRAB transcriptional inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some implementations, the TALEN method can be used to silence or reduce the expression of PD-1 and TIGIT in TIL.

III. Gene Editing Methods

As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) genetically modified via gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing performed via nucleotide insertion (RNA or DNA) into a TIL population to suppress the expression of one or more proteins. Embodiments of the present invention also provide a method for expanding TILs into a therapeutic population, wherein the method includes gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, and these gene editing techniques are suitable for use according to the present invention.

In some embodiments, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes a retroviral transduction step. In one embodiment, the method of genetically modifying a TIL population includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, a method for genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which a transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long-term expression of the transposase does not occur in transgenic cells, for example, a transposase provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems include salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In one embodiment, the method of genetically modifying a TIL population includes the step of stably incorporating a gene for producing or repressing (e.g., silencing) one or more proteins. In one embodiment, the method of genetically modifying a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In another embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating a TIL with a pulsed electric field to alter, manipulate, or induce a defined and controlled permanent or temporary change in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In one embodiment, the electroporation method is a pulsed electroporation method, which includes the step of treating TILs with a pulsed electric field to induce pore formation in the TILs, including the step of applying a series of at least three DC electrical pulses to the TILs, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time and such that the survival rate of the TILs is maintained. In one embodiment, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In one embodiment, the method for genetically modifying a TIL population includes the step of transfection using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

According to one embodiment, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems, such as Cas9, target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA bases and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, a method for amplifying TILs into a therapeutic population may be performed according to any embodiment of the method described herein (e.g., procedure 2A) or as described in WO2018/081473A1, WO 2018/129332 A1, or WO 2018/182817 A1, wherein said method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by comparing gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

1. TALE method

A method for amplifying TILs into a therapeutic population can be made according to any embodiment of the method described herein or as described in WO 2018/081473 A1, WO 2018/129332A1, or WO 2018/182817 A1, wherein the method further includes gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the use of the TALE method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the TALE method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for “transcription activator-like effector” protein, which includes TALEN (“transcription activator-like effector nuclease”). The method of gene editing using the TALE system is also referred to herein as the TALE method. TALE is a naturally occurring protein from the plant pathogenic bacterium *Xanthomonas* and contains a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize continuous DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by 14–20 base-pair spacers, bring FokI monomers closer together for dimerization and to generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and T NFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA , IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

Non-limiting examples of TALE-nucleases targeting the PD-1 gene are provided in the table below. In these examples, the targeted genomic sequence contains two 17-base-pair (bp) long sequences (referred to as half-targets, shown in uppercase letters) separated by a 15-bp spacer. Each half-target is recognized by a repetitive sequence of the half-TALE-nuclease listed in the table. Thus, according to a particular embodiment, the TALE-nuclease according to the invention recognizes and cleaves target sequences selected from the group consisting of: SEQ ID NO:127 and SEQ ID NO:128. TALEN sequences and gene editing methods are also described in Gautron et al., Molecular Therapy: Nucleic Acids, December 2017, Vol. 9: 312-321, which is incorporated herein by reference.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

2. Cas-CLOVER method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs using a Cas-CLOVER method. According to a particular embodiment, the use of the Cas-CLOVER method during the TIL amplification process can cause silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the Cas-CLOVER method during the TIL amplification process can cause enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Cas-CLOVER is a dimerizing, high-fidelity site-specific nuclease (SSN) composed of a fusion of catalytically dead SpCas9 (dCas9) and a nuclease domain from a Clostridium Clo051 type IIs restriction endonuclease (Madison et al., “Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched alloeneic CAR-T cells”, Molecular Therapy-Nucleic Acids, 2022). This produces a nuclease whose activity is determined by the dimerization of the Clo051 nuclease domain, which recognizes two adjacent 20-nt target sequences via RNA guidance. Unlike paired nicking enzyme approaches, for example, when using the Cas9-D10A mutant, monomeric Cas-CLOVER does not introduce nicks or DSBs. Cas-CLOVER has been shown to have low off-target nuclease activity.

Exemplary Cas-CLOVER systems include those described in WO2019/126578, the contents of which are incorporated herein by reference in their entirety. In embodiments, the Cas-CLOVER system comprises a fusion protein containing a DNA-localizing component and an effector molecule, substantially consisting of or consisting of therein.

DNA positioning components

In this embodiment, the DNA targeting component is capable of binding to a specific DNA sequence. In this embodiment, the DNA targeting component is selected from, for example, DNA-binding oligonucleotides, DNA-binding proteins, DNA-binding protein complexes, and combinations thereof. Other suitable DNA-binding components will be recognized by those skilled in the art.

In one embodiment, the DNA targeting component comprises oligonucleotides targeting one or more specific loci in the genome. The oligonucleotides may be selected from DNA, RNA, DNA/RNA hybrids, and combinations thereof.

In some embodiments, the DNA positioning component comprises a nucleotide-binding protein or protein complex that binds an oligonucleotide upon binding to the target DNA. The protein or protein complex may be able to recognize features selected from RNA-DNA heteroduplexes, R-loops, or combinations thereof. In some embodiments, the DNA positioning component comprises a protein or protein complex capable of recognizing an R-loop selected from Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, or combinations thereof. In some embodiments, the DNA positioning component comprises an engineered protein capable of binding to the target DNA. In some embodiments, the DNA positioning component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, transcription activator-like (TAL) arrays, and combinations thereof. In some embodiments, the DNA positioning component comprises a protein containing a naturally occurring DNA-binding domain. In some embodiments, the DNA positioning component comprises a bZIP domain, a helix-loop-helix, a helix-turn-helix, an HMG-box, a leucine zipper, a zinc finger, or combinations thereof. In some embodiments, the DNA positioning component comprises an oligonucleotide targeting a specific locus in the genome. Exemplary oligonucleotides include, but are not limited to, DNA, RNA, DNA/RNA hybrids, and any combination thereof. In some embodiments, the DNA targeting component comprises a protein or protein complex capable of recognizing features selected from RNA-DNA heteroduplexes, R-loops, and any combinations thereof. Exemplary proteins or protein complexes capable of recognizing R-loops include, but are not limited to, Cas9, cascade complexes, RecA, RNase H, RNA polymerase, DNA polymerase, and any combinations thereof. In some embodiments, the protein or protein complex capable of recognizing R-loops comprises Cas9. In some embodiments, the DNA targeting component comprises a protein capable of binding to a DNA sequence selected from a wide range of nucleases, zinc finger arrays, TAL arrays, and any combinations thereof. In some embodiments, the DNA targeting component comprises an oligonucleotide targeting a specific location in the genome and a protein capable of binding to a target DNA sequence.

In one embodiment, the DNA positioning component comprises at least one guide RNA (gRNA), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises two gRNAs, substantially consisting of or consisting of them, wherein the first gRNA specifically binds to the first strand of the double-stranded DNA target sequence, and the second gRNA specifically binds to the second strand of the double-stranded DNA target sequence. Alternatively, in one embodiment, the DNA positioning component comprises a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), substantially consisting of or consisting of it. In another embodiment, the DNA positioning component comprises a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Ralstonia*, substantially consisting of or consisting of it.

Effect molecules

In the implementation scheme, the effector molecule is capable of producing a predetermined effect at a specific locus in the genome. Exemplary effector molecules include, but are not limited to, transcription factors (activators or repressors), chromatin remodeling factors, nucleases, exonucleases, endonucleases, transposases, methyltransferases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, helicases, fluorophores, or any combination thereof.

In some embodiments, the effector molecule comprises a transposase. In some embodiments, the effector molecule comprises PB transposase (PBase). In some embodiments, the effector molecule comprises a nuclease. Non-limiting examples of nucleases include restriction endonucleases, homing endonucleases, S1 nucleases, mung bean nucleases, pancreatic DNase I, micrococcal nucleases, yeast HO endonucleases, or any combination thereof. In some embodiments, the effector molecule comprises a restriction endonuclease. In some embodiments, the effector molecule comprises a type IIS restriction endonuclease. In some embodiments, the effector molecule comprises a nuclease. Non-restrictive examples of endonucleases include AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, and Clo051. In the implementation scheme, the effector molecule includes BmrI, BfiI, or Clo051.

In embodiments, the effector molecule comprises a homodimer or heterodimer, substantially composed of, or composed of. In embodiments, the effector molecule comprises a nuclease, optionally an endonuclease, substantially composed of, or composed of. In embodiments, the effector molecule, including those comprising a homodimer or heterodimer, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially composed of, or composed of. In embodiments, Cas9 is catalytically inactive or “inactivated” Cas9 (dCas9 (SEQ ID NO: 302 and 303 of WO2019/126578)). In embodiments, Cas9 is the catalytically inactive or “inactivated” nuclease domain of Cas9. In embodiments, dCas9 is encoded by a shorter sequence derived from full-length, catalytically inactivated Cas9, referred herein as “small” dCas9 or dSaCas9 (SEQ ID NO: 23 of WO2019/126578).

In embodiments of the fusion protein, the effector molecule comprises, is substantially composed of, or is composed of, one or more type II nucleases, homodimers or heterodimers of type II nucleases. In the implementation scheme, the type II nuclease includes one or more of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises Cas9, a Cas9 nuclease domain, or a fragment thereof that forms a heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, comprises a catalytically inactive form of Cas9 (e.g., dCas9 or dSaCas9) or a fragment thereof that forms a heterodimer with Clo051, and is substantially composed of or constitutes thereof. An exemplary Clo051 nuclease domain may comprise the following amino acid sequence, and is substantially composed of or constitutes thereof:

EGIKSNISLLKDELRGQISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE

KIRSGEMTIEELERAMFNNSEFILKY(SEQ ID NO:238).

In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces*, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, BfiI, or BmrI, and is substantially composed of or constitutes thereof. In embodiments, the effector molecule, including those comprising homodimers or heterodimers, contains a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rochetomyces* that forms a homodimer or heterodimer with Clo051, and is substantially composed of or constitutes thereof.

connect

In some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule, substantially consisting of or consisting of the latter. In some embodiments, nucleic acid sequences encoding one or more components of the fusion protein can be operatively linked, for example, to an expression vector. In some embodiments, the fusion protein is a chimeric protein. In some embodiments, the fusion protein is encoded by one or more recombinant nucleic acid sequences. In some embodiments, the fusion protein further includes a linker region to operatively link the two components of the fusion protein. For example, in some embodiments, the fusion protein comprises a DNA positioning component and an effector molecule operatively linked by a linker region, substantially consisting of or consisting of the latter. In some embodiments, the DNA positioning component, linker region, and effector molecule can be encoded by one or more nucleic acid sequences inserted into an expression cassette and/or expression vector, such that translation of the nucleic acid sequences produces the fusion protein. In some embodiments, the fusion protein can include a non-covalent link between the DNA positioning component and the effector molecule. The non-covalent link can comprise an antibody, antibody fragment, antibody mimic, or scaffold protein.

Fusion protein

In one embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises Cas9, a Cas9 nuclease domain, or a fragment thereof, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated Cas9 (dCas9) or an inactivated nuclease domain, substantially consisting of or consisting of it. In another embodiment, the DNA localization component comprises at least one gRNA, substantially consisting of or consisting of it, and the effector molecule comprises inactivated small Cas9 (dSaCas9), substantially consisting of or consisting of it. In yet another embodiment, the effector molecule comprises Cas9, dCas9, dSaCas9, or a nuclease domain thereof and a second endonuclease, substantially consisting of or consisting of it. The second endonuclease may include, or be substantially composed of, type IIS endonucleases, including but not limited to, AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, and BmgBI. One or more of BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI, or Clo051.

In embodiments of the fusion protein, the DNA localization component comprises, is substantially composed of, or is composed of, a DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also known as a TAL protein), and the effector molecule comprises, substantially composed of, or is composed of, a nuclease. In embodiments of the fusion protein of this disclosure, the DNA localization component comprises, substantially composed of, or is composed of, a DNA-binding domain of a TALEN or TAL protein derived from *Xanthomonas* or *Rhodotorula*, and the effector molecule comprises, substantially composed of, or is composed of, an IIS-type nuclease, including but not limited to AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My One or more of I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

In some embodiments, the exemplary dCas9-Clo051 fusion protein may comprise, substantially consist of, or consist of the amino acid sequence of SEQ ID NO:305 or 307 of WO2019/126578 or the nucleic acid sequence of SEQ ID NO:306 or 308 of WO2019/126578.

Construct

In the embodiments, the nuclease domain comprises dCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises dSaCas9 and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Xanthomonas spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the nuclease domain comprises Rocheborax spp.-TALE and Clo051, substantially composed of or composed of them. In the embodiments, the fusion protein comprises dCas9-Clo051, dSaCas9-Clo051, Xanthomonas spp.-TALE-Clo051, or Rocheborax spp.-TALE-Clo051. In the embodiments, the vector encoding the fusion protein comprises Csy4-T2A-Clo051-G4S linker-dCas9 (Streptococcus pyogenes) or pRT1-Clo051-dCas9 dual NLS.

According to some implementations, the Cas-CLOVER system comprises a fusion protein containing a DNA localization component and an effector molecule, wherein the DNA localization component hybridizes to a target sequence of a DNA molecule in a TIL, wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule, and the effector molecule cleaves the DNA molecule, thereby altering the expression of at least one immune checkpoint molecule.

According to a specific implementation, the Cas-CLOVER method involves silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas-CLOVER system (e.g., dCas9-Clo051, dSaCas9-Clo051, Xanthomonas-TALE-Clo051, or Rochetomyces-TALE-Clo051 fusion protein) that is specific to the target DNA sequence of immune checkpoint genes. The fusion protein can be delivered as DNA, mRNA, or protein. After the genome comes into contact with the Cas-CLOVER system, one or more strands of the target double-stranded DNA can be cleaved. If cleavage occurs in the presence of one or more DNA repair pathways or components thereof, the Cas-CLOVER method interrupts gene expression or modifies the genomic sequence by inserting, deleting, or substituting one or more base pairs. DSBs in cells can be repaired via non-homologous end joining (NHEJ, a mechanism that typically causes insertions or deletions (insertion/deletion) in DNA). Insertions/deletions typically induce frameshifts, resulting in loss-of-function alleles; for example, by inducing premature stop codons within the open reading frame (ORF) of a target gene. According to some implementations, the result is a loss-of-function mutation within a target immune checkpoint gene.

Alternatively, instead of NHEJ, DSB induced by the Cas-CLOVER system can be repaired via homology-directed repair (HDR). Although NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to produce specific nucleotide changes ranging from a single nucleotide change to a large insertion range. According to some implementations, HDR is used to gene edit immune checkpoint genes by delivering a DNA repair template containing the desired sequence to a TIL equipped with a Cas-CLOVER system. The repair template preferably contains the desired edit immediately upstream and downstream of the target gene, as well as other homologous sequences (often referred to as left and right homologous arms).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the Cas-CLOVER method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF1. 0A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, I L6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via the Cas-CLOVER method and which can be used according to embodiments of the invention are described in WO2019126578, US2017/0107541, US2017/0114149, US2018/0187185, and U.S. Patent No. 10,415,024, the contents of which are incorporated herein by reference in their entirety. Resources for performing the Cas-CLOVER method, such as CLOVER mRNA and Cas-CLOVER mRNA constructs, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system regulating the expression of at least one checkpoint protein in multiple cells of a second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culture for about 1 to 3 days to obtain the second TIL population, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a Cas-CLOVER system, the at least one gene editor system inhibiting the expression of at least one checkpoint protein in multiple cells of the second TIL population.

IV. Gene Expression Methods

In some embodiments, the method of genetically modifying TIL populations to regulate protein expression may optionally include a step of stabilizing and incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying TIL populations includes a viral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes a gamma-retroviral transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adenovirus transduction step. In one embodiment, the method of genetically modifying TIL populations includes an adeno-associated virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a herpes simplex virus transduction step. In one embodiment, the method of genetically modifying TIL populations includes a poxvirus transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step, including lentiviral transduction using human immunodeficiency virus (HIV) (including HIV-1). Lentiviral transduction systems and other suitable viral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat’l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71, and U.S. Patents 5,350,674, 5,585,362, and 6,627,442, the disclosures of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16; Hawley et al., Gene Ther. 1994, 1, 136-38; the contents of which are incorporated herein by reference.

1. piggyBac method

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., procedure 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein said methods further include gene editing of at least a portion of the TILs by a piggyBac method (e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases). According to a particular embodiment, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population. Alternatively, the use of the piggyBac method during the TIL amplification process can induce the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population and optionally induce the enhancement of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. In some embodiments, at least one immunomodulatory composition comprises an immunomodulator fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and CD40 agonists (e.g., CD40L or agonistic CD40 binding domains). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and CD40 agonists. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and CD40 agonists.

The piggyBac transposon is a mobile genetic element that efficiently translocates between the donor vector and the host chromosome. This system has virtually no cargo limitations and is completely reversible, leaving no footprint in the genome after excision. The piggyBac transposon/transposase system consists of transposases that recognize piggyBac-specific inverted terminal repeats (ITRs) located flanking the transposon cassette. The transposases excise the transposable element to integrate it into a TT/AA chromosomal locus that preferentially resides in euchromatin regions of the mammalian genome (Ding et al., 2005; and Bradley 2007; Wilson et al., 2007; Wang et al., 2008; Li et al., 2011).

Exemplary piggyBac systems include those described in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac system comprises a transposon/transposase system.

In the implementation scheme, the piggyBac method includes delivering to the TIL a (a) nucleic acid or amino acid sequence containing a sequence encoding a transposase and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon.

In one embodiment, the sequence encoding the transposase is an mRNA sequence. In another embodiment, the sequence encoding the transposase is a DNA sequence. In yet another embodiment, the DNA sequence is a cDNA sequence. In yet another embodiment, the sequence encoding the transposase is an amino acid sequence. The protein Superpiggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA.

Transposons/transposases

Exemplary transposon/transposase systems include, but are not limited to, piggyBac transposon and transposase, Sleeping Beauty transposon and transposase, Helraiser transposon and transposase, and Tol2 transposon and transposase.

The piggyBac transposon recognizes the transposon-specific inverted terminal repeat (ITR) at the end of the transposon and moves the contents between the ITRs to the TTAA chromosomal locus. The piggyBac transposon system has no effective load limitation on the gene of interest that can be included between the ITRs. In embodiments, the transposon is a piggyBac transposon or a piggyBac-like transposon.

Examples of piggyBac and piggyBac-like transposases and transposons include, for example, those disclosed in WO2019/046815, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is overactive. An overactive piggyBac or piggyBac-like transposase is a transposase that is more active than its naturally occurring variants. In embodiments, the overactive piggyBac or piggyBac-like transposase is isolated from or derived from the silkworm (Bombyxmori). A list of overactive amino acid substitutions is available in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety. In embodiments, the piggyBac or piggyBac-like transposase is an integration-deficient type. In embodiments, an integration-deficient piggyBac or piggyBac-like transposase is a transposase capable of cleaving its corresponding transposon but integrating the cleaved transposon at a lower frequency than the corresponding wild-type transposase. A list of defective amino acid substitutions can be found in U.S. Patent No. 10,041,077, the contents of which are incorporated herein by reference in their entirety.

In the embodiments, the piggyBac or piggyBac-like transposon can be inserted into the 5'-TTAT-3 sequence within the target nucleic acid via a piggyBac or piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In the embodiments, and particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or SuperpiggyBac ^™ (SPB) transposase. In the embodiments, and particularly in embodiments where the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

The Sleeping Beauty (SB) transposon is translocated into the target genome via a Sleeping Beauty transposase that recognizes an ITR, and moves the contents between ITRs to the TA chromosomal locus. In an embodiment, the transposon is a Sleeping Beauty transposon. In an embodiment, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In an embodiment, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase.

The Helraiser transposon is translocated by the Helitron transposase. Unlike other transposases, the Helitron transposase does not contain an RNase-H-like catalytic domain, but instead contains a RepHel motif consisting of a replication initiation domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH nuclease superfamily. In embodiments, the transposon is the Helraiser transposon. In embodiments of the Helraiser transposon sequence, the transposase is flanked by left and right terminal sequences, referred to as the LTS and RTS. In embodiments, these sequences terminate with a conserved 5'-TC/CTAG-3' motif. In embodiments, a 19 bp palindromic sequence with the potential to form a hairpin termination structure is located 11 nucleotides upstream of the RTS and contains the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG. In embodiments, and particularly in embodiments where the transposon is the Helraiser transposon, the transposase is the Helitron transposase.

The Tol2 transposon can be isolated from or derived from the genome of the killifish and can be similar to transposons of the hAT family. An exemplary Tol2 transposon of this disclosure is encoded by a sequence comprising approximately 4.7 kilobases and contains a gene encoding a Tol2 transposase, said gene containing four exons. In embodiments, the transposon is a Tol2 transposon. In some embodiments of the methods of this disclosure, and particularly in those embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

In some embodiments, the vector comprises a recombinant and non-naturally occurring DNA sequence encoding a transposon. In some embodiments, the vector comprises DNA of any form, and wherein the vector comprises at least 100 nucleotides (nt), 500 nt, 1000 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6500 nt, 7000 nt, 7500 nt, 8000 nt, 8500 nt, 9000 nt, 9500 nt, 10,000 nt, or any number of nucleotides in between. In some embodiments, the vector comprises single-stranded or double-stranded DNA. In some embodiments, the vector comprises circular DNA. In some embodiments, the vector is a plasmid vector, a nanoplasmid vector, or a small loop. In some embodiments, the vector comprises linear or linearized DNA. In some embodiments, the vector is a double-stranded doggybone ^™ DNA sequence.

In the implementation scheme, the recombinant and non-naturally occurring DNA sequences encoding transposons also include sequences encoding one or more immune checkpoint genes.

In the implementation scheme, the nucleic acid sequence encoding the transposase is a DNA sequence, and the amount of the DNA sequence encoding the transposase and the DNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the DNA sequences encoding transposases and the DNA sequences encoding transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation scheme, the nucleic acid sequence encoding the transposase is an RNA sequence, and the amount of the RNA sequence encoding the transposase and the RNA sequence encoding the transposon is equal to or less than 10.0 μg/100 μL, less than 7.5 μg/100 μL, less than 6.0 μg/100 μL, less than 5.0 μg/100 μL, less than 2.5 μg/100 μL, or less than 1.67 μg/100 μL, less than 0.55 μg/100 μL, less than 0.19 μg/100 μL, or less than 0.10 μg/100 μL in electroporation or nuclear transfection reactions. In some embodiments, the concentrations of the RNA sequences encoding transposases and transposons in the electroporation or nuclear transfection reaction are equal to or less than 100 μg/mL, equal to or less than 75 μg/mL, equal to or less than 60 μg/mL, equal to or less than 50 μg/mL, equal to or less than 25 μg/mL, equal to or less than 16.7 μg/mL, equal to or less than 5.5 μg/mL, equal to or less than 1.9 μg/mL, and equal to or less than 1.0 μg/mL.

In the implementation, the TIL is further modified by a second gene-editing tool, including but not limited to those described herein. In the implementation, the second gene-editing tool may include a piggyBac transposase that is only excised to re-excise the inserted sequence or any portion thereof. For example, a piggyBac transposase that is only excised can be used to "re-excise" a transposon.

According to some embodiments, the piggyBac system comprises a transposon/transposase system, wherein the transposase recognizes ITRs located flanking a transposon cassette containing a cargo encoding one or more immune checkpoint genes, and excises transposable elements to integrate it into a TT/AA chromosomal site, resulting in genomic insertion of the transposon cassette and expression of one or more immune checkpoint genes. According to some embodiments, the cargo encodes two or more immune checkpoint molecules.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the piggyBac method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD), and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using the piggyBac method and which can be used according to embodiments of the invention are described in WO2019/046815, WO2015006700, WO2010085699, WO2010099301, WO2010099296, WO2006122442, WO2001081565, and WO1998040510, the contents of which are incorporated herein by reference in their entirety.

Resources for executing the piggyBac method, such as plasmids for expressing transposons/transposases, are available from companies such as Demeetra and Hera Biolabs.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) Stimulate the second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivering at least one gene editor system comprising a piggyBac system, wherein the at least one gene editor system affects the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population and regulates the expression of at least one checkpoint protein in multiple cells of the second TIL population.

According to some implementation schemes, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(a) Obtaining a first TIL population from a tumor removed from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Add the tumor fragment to the closed system;

(c) A first amplification is performed by culturing the first TIL population for about 3 to 11 days in a cell culture medium containing IL-2 and optionally containing OKT-3 and/or 4-1BB agonist antibodies to generate a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area.

(d) The second TIL population is obtained by stimulating it with OKT-3 and culturing it for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Perform sterile electroporation on the second TIL population to transfer at least one gene editor into multiple cells in the second TIL population;

(f) Allow the second TIL population to rest for approximately 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody and antigen-presenting cells (APC) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the transition from step (f) to step (g) is performed without opening the system;

(h) Collect the therapeutic TIL population obtained from step (g) to provide a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population.

(i) transferring the collected TIL population into an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system; and

(j) Optionally, cryopreservation medium may be used to cryopreserve the collected TIL population.

The electroporation step includes delivery of at least one gene editor system comprising the piggyBac system, said gene editor system influencing the expression of at least one immunomodulatory composition on the cell surface of multiple cells in a second TIL population. In some embodiments, the at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

In some embodiments, methods for genetically modifying TIL populations include using non-viral techniques, such as the piggybac method (e.g., piggybac transposons and transposases or piggybac-like transposons and transposases). In some embodiments, the method includes delivering to the TIL: (a) a nucleic acid or amino acid sequence containing a sequence encoding a transposase; and (b) a recombinant and non-naturally occurring DNA sequence containing a DNA sequence encoding a transposon. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an mRNA sequence. The mRNA sequence encoding the transposase can be generated in vitro. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is a DNA sequence. The DNA sequence encoding the transposase can be generated in vitro. The DNA sequence may be a cDNA sequence. In some embodiments of the methods disclosed herein, the sequence encoding the transposase is an amino acid sequence. The amino acid sequence encoding the transposase can be generated in vitro. A protein super piggybac transposase (SPB) can be delivered after pre-incubation with transposon DNA. In some embodiments, the transposon is a piggybac transposon or a piggybac-like transposon. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac transposase. In some embodiments, and particularly those in which the transposon is a piggyBac-like transposon, the transposase is a piggyBac-like transposase. In some embodiments, the piggyBac transposase comprises the amino acid sequence containing SEQ ID NO:14487 of WO2019046815. In some embodiments, and particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac ^™ or Super piggyBac ^™ (SPB) transposase. In some embodiments, and particularly those in which the transposase is a Super piggyBac ^™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence. In some embodiments of the methods disclosed herein, the transposase is a piggyBac ^™ (PB) transposase. piggyBac (PB) transposases may contain or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof identical to the following sequences:

In some embodiments of the methods disclosed herein, the transposon is a Sleeping Beauty transposon. In some embodiments of the methods disclosed herein, the transposase is a Sleeping Beauty transposase (see, for example, U.S. Patent No. 9,228,180, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase. In some embodiments, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween the following sequences:

In some embodiments, including those in which the Sleeping Beauty transposase is an overactive Sleeping Beauty (SB100X) transposase, the Sleeping Beauty transposase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage thereof, identity with the following sequences:

V.Gen 2TIL Manufacturing Process

Figures 1 and 2 depict an exemplary TIL process family called Gen 2 (also known as process 2A) containing some of these features. An implementation of Gen 2 is shown in Figure 2.

As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs to enhance their metabolic activity and thus improve relative health status prior to transplantation into a patient, as well as methods for testing said metabolic health status. As generally outlined herein, TILs are typically obtained from patient samples and manipulated to amplify their number prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below.

In some implementations, TILs can be cryopreserved. After thawing, they can also be restimulated before being infused into the patient to increase their metabolism.

In some embodiments, the first amplification (including a process called pre-REP and the process shown as step B in Figures 1 and 36) is shortened to 3 to 14 days, and the second amplification (including a process called REP and the process shown as step D in Figure 1 or 36) is shortened to 7 to 14 days, as discussed in detail below, in the embodiments, and in the accompanying drawings. In some embodiments, the first amplification (e.g., the amplification described as step B in Figure 1 or 36) is shortened to 11 days, and the second amplification (e.g., the amplification described as step D in Figure 1 or 36) is shortened to 11 days. In some embodiments, the combination of the first and second amplifications (e.g., the amplification described as steps B and D in Figure 1 or 36) is shortened to 22 days, as discussed in detail below, in the embodiments, and in the accompanying drawings.

The "steps" denoted as A, B, C, etc. in the following text refer to Figure 1 or Figure 36 and certain embodiments described herein. The order of the steps in the following text and Figures 1 and 36 is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, repetitions of steps, and/or omissions of steps.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples and then expanded into larger populations for further manipulation as described herein, optionally cryopreservation, restimulation as outlined herein, and optionally evaluation of phenotypic and metabolic parameters as indicators of TIL health status.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multifocal sampling is used. In some embodiments, surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells include multifocal sampling (i.e., obtaining samples from one or more tumor sites and/or locations in a patient, as well as from one or more tumors at the same location or closely adjacent to each other). Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be lung tissue. In some embodiments, the applicable TIL is obtained from non-small cell lung cancer (NSCLC). Solid tumors can be skin tissue. In some embodiments, the applicable TIL is obtained from melanoma.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as HBSS.

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is a 10X working stock solution at a concentration of 100 mg/mL.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is a 10X working stock solution of 10,000 IU/mL.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is a 10X working stock solution at a concentration of 10 mg/mL.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatic tumor digests and tumor fragments obtained by digesting or fragmenting tumor samples obtained from patients.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as shown in Figure 1 or Figure 36), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container for first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor is 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor is 2mm × 2mm × 2mm. In some embodiments, the tumor is 3mm × 3mm × 3mm. In some embodiments, the tumor is 4mm × 4mm × 4mm.

In some embodiments, the tumor is removed to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of necrotic tissue in each mass. In some embodiments, the tumor is removed to minimize the amount of adipose tissue in each mass.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be used to remove these cells.

In some implementations, the cell suspension collected prior to the first amplification step is referred to as the “primary cell population” or “freshly collected” cell population.

In some embodiments, the cells may optionally be frozen after sample collection and cryopreserved before proceeding to the amplification described in step B (which is described in further detail below and in examples in Figures 1 and 36 and Figure 8).

1. Pleural effusion T cells and TIL

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may originate from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may originate from secondary metastatic cancer cells originating from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural transudate. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments where the disclosed methods utilize pleural fluid, ascites fluid or other cystic fluids containing TILs can be used to perform the same methods to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to further processing steps. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to further processing steps. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of viable TILs. If retained in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in further processing steps of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

B. Step B: First Amplification

In some embodiments, the method of the present invention achieves the acquisition of young TILs, which, compared to older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to the subject/patient), enable increased replication cycles at the time of administration to the subject/patient and thus provide additional therapeutic benefits. Characteristics of young TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32: 415-423; Robbins et al., J. Immunol. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, which are each incorporated herein by reference.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (connection region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 1 or Figure 36. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method referred to as Process 1C, as exemplified in Figures 5 and/or 6. In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased.

Following the dissection or digestion of the tumor fragment as described in step A of, for example, Figure 1 or Figure 36, the resulting cells are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 3 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 7 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells. In some embodiments, this primary cell population is cultured for about 11 days, yielding a primary TIL population typically of about 1 × ^10⁸ main TIL cells.

In some implementations, TIL amplification may be performed using the initial host TIL amplification steps described below and herein (e.g., the steps described in step B of Figure 1 or Figure 36, which may include a process called pre-REP), followed by a second amplification step described below and herein (step D, including a process called rapid amplification protocol (REP) step), followed by optional cryopreservation, and then a second step D described below and herein (including a process called restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic traits and metabolic parameters as described herein.

In TIL culture, the culture is initiated in a 24-well plate, such as using a Costar 24-well cell culture cluster, with a flat bottom (Corning Incorporated, Corning, NY). Each well is seeded with 2 mL of complete culture medium (CM) containing 1 × ^10⁶ tumor digested cells or one tumor fragment and IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digestion cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plate are incubated in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days.

After preparing the tumor fragment, the resulting cells (i.e., the fragment) are cultured in serum containing IL-2 under conditions favorable to TIL growth relative to the tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum (or, in some cases, as outlined herein, in the presence of an APC cell population) and 6000 IU/mL IL-2. This primary cell population is cultured for several days, typically 10 to 14 days, yielding a primary TIL population typically of about 1 × ^10⁸ primary TIL cells. In some embodiments, the growth medium contains IL-2 or a variant thereof during the initial expansion. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20-30 × ^10⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains approximately 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains IL-2 at concentrations between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL.

In some embodiments, the first amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the first amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the cell culture medium contains an anti-CD3 agonist antibody, such as OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, OKT-3 is present in the cell culture medium at the start of the first amplification (day 0). In some embodiments, OKT-3 is added to the cell culture medium at any time during the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 1 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 2 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 3 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 4 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 5 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 6 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 7 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 8 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 9 of the first amplification. In some embodiments, OKT-3 is added to the cell culture medium on day 10 of the first amplification. In some implementations, OKT-3 is added to the cell culture medium on day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 1 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 2 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 3 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 3 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 6 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 7 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 8 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 9 and day 11 of the first amplification. In some implementations, OKT-3 is added once or more between day 10 and day 11 of the first amplification.

In some implementations, OKT-3 is added once or more between day 0 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 0 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 1 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 1 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 2 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 2 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 3 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 5 of the first amplification. In some implementations, OKT-3 is added once or more between day 3 and day 4 of the first amplification.

In some implementations, OKT-3 is added once or more between day 4 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 6 of the first amplification. In some implementations, OKT-3 is added once or more between day 4 and day 5 of the first amplification.

In some implementations, OKT-3 is added once or more between day 5 and day 7 of the first amplification. In some implementations, OKT-3 is added once or more between day 5 and day 6 of the first amplification.

In some implementations, OKT-3 is added once or more between day 6 and day 7 of the first amplification.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In embodiments where culture is initiated in a permeable culture flask with a 40 mL capacity and a 10 ^cm² permeable silica bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10-40 mL of CM containing 10-40 × ^10⁶ live tumor digest cells or 5-30 tumor fragments with IL-2. Both the G-REX10 and the 24-well plates are cultured in a humidified incubator at 37°C and 5% _CO₂ , and after 5 days of culture initiation, half of the culture medium is removed and replaced with fresh CM and IL-2, and after day 5, half of the culture medium is replaced every 2-3 days. In some embodiments, CM is CM1 as described in the examples, see Example 1. In some embodiments, the first amplification is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 3-14 days, as discussed in the embodiments and figures. In some embodiments, the first amplification (including processes such as those described in step B of FIG. 1 or FIG. 36, which may include a process sometimes referred to as pre-REP) is shortened to 7 to 14 days, as discussed in the embodiments and shown in FIG. 4 and FIG. 5, and includes amplification such as that described in step B of FIG. 1 or FIG. 36. In some embodiments, the first amplification in step B is shortened to 10-14 days. In some embodiments, the first amplification is shortened to 11 days, as discussed in the amplification described in step B of FIG. 1 or FIG. 36.

In some embodiments, the first TIL amplification can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the first TIL amplification can be performed for 1 to 14 days. In some embodiments, the first TIL amplification can be performed for 2 to 14 days. In some embodiments, the first TIL amplification can be performed for 3 to 14 days. In some embodiments, the first TIL amplification can be performed for 4 to 14 days. In some embodiments, the first TIL amplification can be performed for 5 to 14 days. In some embodiments, the first TIL amplification can be performed for 6 to 14 days. In some embodiments, the first TIL amplification can be performed for 7 to 14 days. In some embodiments, the first TIL amplification can be performed for 8 to 14 days. In some embodiments, the first TIL amplification can be performed for 9 to 14 days. In some embodiments, the first TIL amplification can be performed for 10 to 14 days. In some embodiments, the first TIL amplification can be performed for 11 to 14 days. In some embodiments, the first TIL amplification can proceed for 12 to 14 days. In some embodiments, the first TIL amplification can proceed for 13 to 14 days. In some embodiments, the first TIL amplification can proceed for 14 days. In some embodiments, the first TIL amplification can proceed for 1 to 11 days. In some embodiments, the first TIL amplification can proceed for 2 to 11 days. In some embodiments, the first TIL amplification can proceed for 3 to 11 days. In some embodiments, the first TIL amplification can proceed for 4 to 11 days. In some embodiments, the first TIL amplification can proceed for 5 to 11 days. In some embodiments, the first TIL amplification can proceed for 6 to 11 days. In some embodiments, the first TIL amplification can proceed for 7 to 11 days. In some embodiments, the first TIL amplification can proceed for 8 to 11 days. In some embodiments, the first TIL amplification can proceed for 9 to 11 days. In some embodiments, the first TIL amplification can proceed for 10 to 11 days. In some embodiments, the first TIL amplification can proceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the first amplification period. In some embodiments, the first amplification period, including, for example, during step B of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, as discussed in the examples and figures, the first amplification process (including a process called pre-REP; for example, step B according to Figure 1 or Figure 36) is shortened to 3 to 14 days. In some embodiments, the first amplification in step B is shortened to 7 to 14 days. In some embodiments, the first amplification in step B is shortened to 10 to 14 days. In some embodiments, the first amplification is shortened to 11 days.

In some embodiments, the first amplification is performed in a closed-system bioreactor, such as step B according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

1. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Transition from the first amplification to the second amplification

In some cases, the host TIL population obtained from the first amplification, including, for example, the TIL population obtained from step B as shown in Figure 1 or Figure 36, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first amplification (referred to as the second TIL population) can undergo a second amplification (which may include an amplification sometimes referred to as REP) and then be cryopreserved as discussed below. Similarly, where the genetically modified TILs are intended for therapeutic use, the first TIL population (sometimes referred to as the host TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be genetically modified before amplification or after the first amplification and before the second amplification for use in a suitable treatment.

In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is stored until a phenotypic analysis for selection is performed. In some embodiments, the TIL obtained from the first amplification (e.g., from step B as indicated in Figure 1 or Figure 36) is not stored and proceeds directly to the second amplification. In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 10 days after fragmentation. In some implementations, the transition from the first amplification to the second amplification occurs approximately 7 to 14 days after fragmentation. In other implementations, the transition from the first amplification to the second amplification occurs approximately 14 days after fragmentation.

In some embodiments, the transition from the first amplification to the second amplification occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 1 to 14 days after fragmentation. In some embodiments, the first TIL amplification may occur 2 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 5 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 7 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after fragmentation. In some embodiments, the transition occurs 9 to 14 days after fragmentation. In some embodiments, the transition occurs 10 to 14 days after fragmentation. In some embodiments, the transition occurs 11 to 14 days after fragmentation. In some embodiments, the transition occurs 12 to 14 days after fragmentation. In some embodiments, the transition occurs 13 to 14 days after fragmentation. In some embodiments, the transition occurs 14 days after fragmentation. In some embodiments, the transition occurs 1 to 11 days after fragmentation. In some embodiments, the transition occurs 2 to 11 days after fragmentation. In some embodiments, the transition occurs 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 11 days after fragmentation. In some embodiments, the transition occurs 5 to 11 days after fragmentation. In some embodiments, the transition occurs 6 to 11 days after fragmentation. In some embodiments, the transition occurs 7 to 11 days after fragmentation. In some embodiments, the transition occurs 8 to 11 days after fragmentation. In some embodiments, the transition occurs 9 to 11 days after fragmentation. In some embodiments, the transition occurs 10 to 11 days after fragmentation. In some embodiments, the transition occurs 11 days after fragmentation.

In some embodiments, TILs are not stored after the first amplification and before the rapid second amplification, and the TILs undergo the second amplification directly (e.g., in some embodiments, no storage occurs during the transition from steps B to D as shown in Figure 1 or Figure 36). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from the first amplification (the second TIL population) undergo the second amplification directly without a transition period.

In some embodiments, the transition from the first amplification to the second amplification (e.g., step C according to Figure 1 or Figure 36) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 2-7 days, approximately 3-7 days, approximately 4-7 days, approximately 5-7 days, approximately 6-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

According to this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from MiltenyiBiotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulation of TIGIT expression and downregulation of one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing step also includes a quiescent step. According to some implementations, the quiescent step includes incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Second amplification

In some embodiments, the number of TIL cell populations is expanded after collection and initial host treatment, for example, after steps A and B and a transition referred to as step C (as indicated in Figure 1 or Figure 36). This further expansion is referred to herein as a second expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP); and the process indicated in step D of Figure 1 or Figure 36. The second expansion is typically performed in a breathable container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 1 or Figure 36) may be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL amplification may be performed for about 7 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 8 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 9 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 10 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 11 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 12 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 13 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 14 days.

In some embodiments, the second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the procedures indicated in step D of Figure 1 or Figure 36). For example, TILs can be rapidly amplified using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as about 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (available from BioLegend, San Diego, CA). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation to the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of: urinumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, the second amplification period, including, for example, during step D of the process according to FIG. 1 or FIG. 36 and as described herein, may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step D of the process according to FIG. 1 or FIG. 36 and as described herein.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, during rapid expansion and/or secondary expansion, the ratio of TILs to PBMCs and/or antigen-presenting cells is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and/or secondary expansion is between 1:100 and 1:200.

In some implementations, REP and/or secondary amplification are performed in culture flasks, where the host TIL is mixed with 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. The medium is replaced (typically 2/3 replaced with fresh medium via aspiration) until the cells are transferred to a replacement growth chamber. The replacement growth chamber comprises G-REX culture flasks and aeration containers, as discussed more fully below.

In some embodiments, the second amplification (which may include a process referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second amplification is shortened to 11 days.

In some embodiments, the REP and/or second amplification can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or aeration dishes (G-REX culture flasks). In some embodiments, the second amplification (including amplification referred to as rapid amplification) is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs suspended in 150 mL of medium can be added to each T-175 culture flask. The TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The T-175 culture flasks can be incubated at 37°C and 5% _CO₂ . Half of the medium can be replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some implementations, on day 7, cells from two T-175 culture flasks can be combined in a 3L bag, and 300 mL of AIM V with 5% human AB serum and 3000 IU/mL IL-2 can be added to 300 mL of TIL suspension. The cell count in each bag is counted daily or every two days, and fresh culture medium is added to maintain the cell count between 0.5 and 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (which may include an amplification referred to as REP, as well as the amplification mentioned in step D of Figure 1 or Figure 36) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing, New Brighton, MN, USA) with a 100 cm aerated silica bottom. 5 × ^10⁶ or 10 × ^10⁶ TILs can be cultured together with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). The G-REX-100 culture flask can be incubated at 37°C in 5% _CO₂ . On day 5, 250 mL of the supernatant can be removed and transferred to a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. TIL precipitate can be resuspended in 150 mL of fresh culture medium containing 5% human AB serum and 3000 IU/mL IL-2, and added back to the initial G-REX-100 culture flask. When TILs are serially amplified in G-REX-100 culture flasks, on day 7, TILs from each G-REX-100 flask can be resuspended in 300 mL of culture medium present in each flask, and the cell suspension can be aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 can be added to each culture flask. The G-REX-100 culture flasks can be incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 can be added to each G-REX-100 culture flask. Cells can be harvested on day 14 of culture. In some implementations, the process uses different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different number of repetitions.

In some embodiments, the second amplification (including an amplification termed REP) is performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some embodiments, two-thirds of the medium is replaced with fresh medium by aspiration. In some embodiments, the alternative growth chamber comprises a G-REX culture flask and a ventilated container, as discussed more fully below.

In some embodiments, a second amplification (including an amplification referred to as REP) is performed, and the process also includes a step of selecting the TIL for superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability assays may be performed after a second amplification (including amplification known as REP amplification) using standard assays known in the art. For example, a trypan blue exclusion assay may be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample may be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, the second amplification of TILs (including amplification referred to as REP) can be performed using T-175 culture flasks and aeration bags as previously described (Tran et al., 2008, Jimmunother., 31, 742-751, and Dudley et al., 2003, Jimmunother., 26, 332-342) or aeration G-REX culture flasks. In some embodiments, the second amplification is performed using culture flasks. In some embodiments, the second amplification is performed using aeration G-REX culture flasks. In some embodiments, the second amplification is performed in T-175 culture flasks, and approximately 1 × ^10⁶ TILs are suspended in approximately 150 mL of culture medium and added to each T-175 culture flask. TILs were cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs used as "feeder" cells, and the cells were cultured in a 1:1 mixture (50/50 medium) of CM and AIM-V supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. T-175 culture flasks were incubated at 37°C and 5% _CO2 . In some embodiments, half of the medium was replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 culture flasks were pooled in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 was added to 300 mL of TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh culture medium can be added to maintain the cell count between approximately 0.5 and approximately 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (including an amplification referred to as REP) is performed in a 500 mL volumetric flask (G-REX-100, Wilson Wolf) with a 100 ^cm² permeable silica bottom. Approximately 5 × ^10⁶ or 10 × ^10⁶ TILs are incubated with irradiated allogeneic PBMCs at a 1:100 ratio in 400 mL of 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The G-REX-100 flask is incubated at 37°C and 5% _CO₂ . In some embodiments, on day 5, 250 mL of the supernatant is removed and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL precipitate is then resuspended in 150 mL of fresh 50/50 medium with 3000 IU/mL IL-2 and added back to the initial G-REX-100 flask. In the embodiment of continuous TIL amplification in G-REX-100 culture flasks, on day 7, the TIL from each G-REX-100 flask is resuspended in 300 mL of culture medium present in each flask, and the cell suspension is aliquoted into three 100 mL aliquots suitable for inoculating three G-REX-100 culture flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 is added to each culture flask. The G-REX-100 culture flasks are incubated at 37°C and 5% _CO2 , and after 4 days, 150 mL of AIM-V containing 3000 IU/mL IL-2 is added to each G-REX-100 culture flask. Cells are harvested on day 14 of culture. In some embodiments, the process is performed using different centrifugation speeds (400 g, 300 g, 200 g, for 5 minutes) and different number of repetitions.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some implementations, the second amplification medium (e.g., sometimes referred to as CM2 or second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the second amplification is performed in a closed-system bioreactor, such as step D according to Figure 1 or Figure 36. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve vertical scaling up of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve lateral scaling out of the culture by: (a) performing rapid or second amplification by culturing TILs in a first small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the TILs transferred from the first small-scale culture is cultured in a second small-scale culture for approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 3 to 7 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid or second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture for approximately 5 days in a first container (e.g., a G-REX-100MCS container); and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid or second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid or second amplification is performed for a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid or second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid or second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid or secondary amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid or secondary amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains the same components as the cell culture medium used for rapid or secondary expansion after splitting. In some embodiments, the cell culture medium used for rapid or secondary expansion before splitting contains different components than the cell culture medium used for rapid or secondary expansion after splitting.

In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid or secondary expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

1. Feeder cells and antigen-presenting cells

In some embodiments, the second amplification procedure described herein (e.g., including the amplification described in step D of Figure 1 or Figure 36, and an amplification referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells placed into the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some implementations, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 50 × ^10⁶ TILs. In still other embodiments, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 25 × ^10⁶ TILs.

In some embodiments, the second amplification procedure described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs in the second expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is possible to perform rapid and/or secondary expansion of TILs using combinations of cytokines and two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796A1, the disclosure of which is incorporated herein by reference.

Step E: Collect TIL

Cells can be collected after the second amplification step. In some embodiments, TILs are collected after one, two, three, four, or more amplification steps, as shown in, for example, Figure 1 or Figure 36. In some embodiments, TILs are collected after two amplification steps, as shown in, for example, Figure 1 or Figure 36.

TILs can be collected by any suitable and aseptic method, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the methods of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, collection, such as step E according to Figure 1 or Figure 36, is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, step E according to Figure 1 or Figure 36 is performed according to the process described herein. In some embodiments, the closed system is introduced under aseptic conditions via a syringe to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is employed.

In some embodiments, TIL is collected according to the methods described in the embodiments. In some embodiments, TIL between day 1 and day 11 is collected using the methods described in the steps mentioned herein, such as the day 11 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 24 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments. In some embodiments, TIL between day 12 and day 22 is collected using the methods described in the steps mentioned herein, such as the day 22 TIL collection in the embodiments.

Step F: Final preparation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 1 or Figure 36 and outlined in detail above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, TILs amplified using the APCs of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using the PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VI. Gen 3TIL Manufacturing Process

Without being limited by any particular theory, it is believed that the initiation of a first expansion to initiate T cell activation, followed by a rapid second expansion to enhance T cell activation, as described in the method of the present invention, allows for the preparation of expanded T cells that retain a "younger" phenotype, and therefore it is anticipated that the expanded T cells of the present invention will exhibit higher cytotoxicity against cancer cells compared to T cells expanded by other methods. In particular, it is believed that initiating T cell activation by exposure to anti-CD3 antibodies (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), as taught in the method of the present invention, and then enhancing it by subsequent exposure to additional anti-CD3 antibodies (e.g., OKT-3), IL-2, and APCs, limits or avoids the maturation of T cells in the culture medium, thereby producing a population of T cells with a less mature phenotype, which are less exhausted by culture expansion and exhibit higher cytotoxicity against cancer cells. In some implementations, the rapid second expansion step is broken down into multiple steps to achieve vertical scaling up of culture scale by: (a) performing rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the T cells from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container and culturing the T cells from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the T cells transferred from the first small-scale culture is cultured in the second small-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the T cells from the small-scale culture to 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the T cells transferred from the first small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs. In some implementations, after the rapid amplification split, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some implementations, after rapid expansion, multiple subpopulations contain a therapeutically effective amount of TIL. In some implementations, after rapid expansion, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some implementations, after rapid expansion, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, rapid amplification is performed over a period of approximately 1 to 5 days before being split into multiple steps. In some implementations, the splitting of rapid amplification occurs approximately day 1, day 2, day 3, day 4, or day 5 after the start of rapid amplification.

In some implementations, the split of rapid amplification occurs approximately day 8, 9, 10, 11, 12, or 13 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of rapid amplification occurs approximately day 10 after the start of the first amplification. In another exemplary implementation, the split of rapid amplification occurs approximately day 11 after the start of the first amplification.

In some implementations, rapid amplification is performed for a period of approximately 4 to 11 days after the split. In some implementations, rapid amplification is performed for a period of approximately 3, 4, 5, 6, 7, 8, 9, 10, or 11 days after the split.

In some embodiments, the cell culture medium used for rapid expansion before splitting contains the same components as the cell culture medium used for rapid expansion after splitting. In some embodiments, the cell culture medium used for rapid expansion before splitting contains different components than the cell culture medium used for rapid expansion after splitting.

In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion prior to cleavage comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by supplementing the cell culture medium in the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is prepared by replacing the cell culture medium in the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after splitting is prepared by replacing the cell culture medium used for rapid expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

In some implementations, the rapid amplification split is performed in a closed system.

In some embodiments, vertical scaling of the TIL culture during rapid expansion includes adding fresh cell culture medium to the TIL culture (also known as feeding the TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, the fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system, such as the Xuri W25.

In some implementations, rapid second expansion is performed after the T cell activation achieved by initiating the first expansion begins to decrease, slow down, decline, or subside.

In some cases, rapid second expansion occurs when T cell activation, achieved through initiating the first expansion, has decreased by just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, or 47%. The percentages are 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 100%.

In some implementations, the rapid second expansion is performed after the T-cell activation achieved by initiating the first expansion has decreased by just or about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

In some implementations, rapid second expansion occurs when T cell activation achieved by initiating the first expansion has decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46%. The percentages are 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after that.

In some cases, rapid second expansion occurs when T cell activation achieved through initiating first expansion has decreased to at most just or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%. %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% after that.

In some implementations, the reduction in T cell activation achieved by initiating the first expansion is determined by a decrease in the amount of interferon-γ released by T cells in response to antigen stimulation.

In some implementations, the initial expansion of T cells occurs within a period of at most exactly 7 days or about 8 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or approximately 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, the initial expansion of T cells is carried out over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

In some implementations, rapid secondary expansion of T cells takes place over a period of at most exactly or about 11 days.

In some implementations, rapid secondary expansion of T cells is carried out within a period of at most exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, rapid secondary expansion of T cells is carried out over time periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 11 days.

In some implementations, the initial expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells is carried out within a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some implementations, the initial expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 8 days, and the rapid second expansion of T cells occurs within a period of exactly or approximately 1 day to exactly or approximately 9 days.

In some implementations, the initial expansion of T cells occurs over an 8-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, the initial expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs within a period of exactly or about 1 day to exactly or about 9 days.

In some implementations, the initial expansion of T cells occurs over a 7-day period, and the rapid second expansion of T cells occurs over a 9-day period.

In some implementations, T cells are tumor-infiltrating lymphocytes (TILs).

In some implementations, the T cells are bone marrow infiltrating lymphocytes (MIL).

In some implementations, the T cells are peripheral blood lymphocytes (PBLs).

In some implementations, the T cells are obtained from a donor with cancer.

In some implementations, T cells are TILs obtained from tumors removed from patients with cancer.

In some implementations, the T cells are MILs obtained from the bone marrow of patients with hematological malignancies.

In some implementations, the T cells are peripheral blood mononuclear cells (PBMCs) derived from a donor. In some implementations, the donor has cancer. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some implementations, the donor has a tumor. In some implementations, the tumor is a liquid tumor. In some implementations, the tumor is a solid tumor. In some implementations, the donor has a hematological malignancy.

In some aspects of this disclosure, immune effector cells (e.g., T cells) can be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL separation). In a preferred aspect, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected via apheresis may be washed to remove plasma fractions and optionally placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations. In one aspect, T cells are separated from peripheral blood lymphocytes by lysing erythrocytes and, for example, by depleting monocytes through PERCOLL gradient centrifugation or by countercurrent centrifugation.

In some embodiments, the T cells are PBLs isolated from whole blood or lymphocyte-rich apheresis products from a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus (HPV), head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematologic malignancy. In some embodiments, PBL is isolated from whole blood or lymphocyte-rich apheresis products using a positive or negative selection method, i.e., removing PBL using markers of the T-cell phenotype (e.g., CD3+CD45+), or removing non-T-cell phenotype cells while leaving PBL. In other embodiments, PBL is separated by gradient centrifugation. After PBL is isolated from donor tissue, the initial amplification of PBL can be initiated according to the initial amplification step of any method described herein by seeding a suitable number of isolated PBLs (approximately 1 × ^10⁷ PBLs in some embodiments) into an initial amplification culture.

An exemplary TIL process containing some of these features, referred to as process 3 (also referred to herein as Gen 3), is depicted in FIG8 (in particular, for example, FIG8B and/or FIG8C and/or FIG8D) and FIG36, and some advantages of this embodiment of the invention compared to Gen 2 are described in FIG1, FIG2, FIG8, FIG30 and FIG31 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). Implementations of Gen 3 are shown in Figures 1, 8, 30, and 36 (particularly, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). Process 2A or Gen 2 is also described in U.S. Patent Publication No. 2018/0280436, which is incorporated herein by reference in its entirety. The Gen 3 process is also described in International Patent Publication WO 2020/096988.

As discussed and generally summarized herein, TILs are derived from patient samples and manipulated using a TIL amplification process described herein and referred to as Gen 3 to amplify their number before transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as discussed below. In some embodiments, TILs may be cryopreserved before or after amplification. After thawing, they may also be restimulated to increase their metabolism before infusion into a patient.

In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, and the rapid second amplification... The amplification process (including the process referred to herein as the Rapid Amplification Protocol (REP) and the process shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 8 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 7 days, and the rapid second amplification... The amplification process (including the procedure referred to herein as the Rapid Amplification Protocol (REP) and the procedure shown as step D in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is shortened to 1 to 9 days, as discussed in detail below, in the examples, and in the accompanying drawings. In some implementations, the initiation of the first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in Figure 8 (in particular, e.g., Figure 8B and/or Figure 8C)) takes 1 to 7 days, and the rapid second amplification (including the process referred herein as rapid amplification protocol (REP), and the process shown as step D in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) takes 1 to 10 days, as discussed in detail below and in the embodiments and figures. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 9 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 8 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) takes 9 to 10 days. In some implementations, the initiation of the first amplification (e.g., the amplification described as step B in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is 7 to 9 days. In some embodiments, the combination of initiating the first amplification and rapid second amplification (e.g., amplification described in Figure 8 (particularly, e.g., Figure 8B and/or Figure 8C as steps B and D) lasts 14-16 days, as discussed in detail below and in the examples and figures. In particular, it is believed that certain embodiments of the invention include an initiating first amplification step, wherein TILs are activated by exposure to an anti-CD3 antibody (e.g., OKT-3) in the presence of IL-2 or by exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody (e.g., OKT-3). In some embodiments, the TILs activated in the initiating first amplification step as described above are a first TIL population, i.e., a primary cell population.

The “step” designations A, B, C, etc. in the following text are non-limiting examples with reference to Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and certain non-limiting embodiments described herein. The order of steps in the following text and Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) is exemplary, and the methods disclosed in this application and herein cover any combination or order of steps, as well as additional steps, step repetitions, and/or step omissions.

Step A: Obtaining a tumor sample from the patient.

Generally, TILs are initially obtained from patient tumor samples (“primary TILs”) or from circulating lymphocytes (e.g., peripheral blood lymphocytes, including peripheral blood lymphocytes with TIL-like characteristics), and are then expanded into larger populations for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotype and metabolic parameters as indicators of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be any type of cancer, including (but not limited to) breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including (but not limited to) squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the applicable TIL is obtained from malignant melanoma tumors because reports indicate that these tumors have particularly high levels of TIL.

Once obtained, tumor samples are typically fragmented using a sharpening device into small flakes ranging from 1 ^mm³ to approximately 8 ^mm³ , with approximately 2–3 ^mm³ being particularly suitable. TILs are cultured from these fragments using enzymatically digested tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium and mechanically dissociating it for approximately 1 minute, followed by incubation at 37°C in 5% _CO₂ for 30 minutes, and then repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for amplifying TILs or for treating cancer.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociating enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a certain amount of sterile buffer (e.g., HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution is variable and may require determination of its concentration. In some embodiments, the concentration of the lyophilized stock solution may be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises approximately 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

As noted above, in some embodiments, the tumor-in-lab (TIL) is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is enzymatically digested as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C and 5% _CO2 . In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 under rotation in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours in an enzyme mixture containing collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested for 1-2 hours at 37°C and 5% _CO2 in an enzyme mixture containing collagenase, DNase, and neutral protease under _rotation . In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% _CO2 under constant rotation. In some embodiments, the whole tumor is combined with the enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of the collagenase is 100 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 1000 IU/mL DNase and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally, cell suspensions obtained from tumors are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

In some embodiments, fragmentation includes physical fragmentation, including, for example, splitting and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is splitting. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient. In some embodiments, TILs may initially be cultured from enzymatically digested tumor fragments and tumor fragments obtained from a patient.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as provided in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), the tumor is physically fragmented. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the tumor is fragmented and 40 fragments or blocks are placed in each container to initiate the first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the tumor intraepithelial membrane (TIL) is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharpsegmentation. In some embodiments, the tumor fragments are between about 1 ^mm³ and 10 ^mm³ . In some embodiments, the tumor fragments are between about 1 ^mm³ and 8 ^mm³ . In some embodiments, the tumor fragments are about 1 ^mm³ . In some embodiments, the tumor fragments are about 2 ^mm³ . In some embodiments, the tumor fragments are about 3 ^mm³ . In some embodiments, the tumor fragments are about 4 ^mm³ . In some embodiments, the tumor fragments are about 5 ^mm³ . In some embodiments, the tumor fragments are about 6 ^mm³ . In some embodiments, the tumor fragments are about 7 ^mm³ . In some embodiments, the tumor fragments are about 8 ^mm³ . In some embodiments, the tumor fragments are about 9 ^mm³ . In some embodiments, the tumor fragments are about 10 ^mm³ . In some embodiments, the tumor fragments are 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor fragments are 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2mm × 2mm × 2mm. In some embodiments, the tumor fragment is 3mm × 3mm × 3mm. In some embodiments, the tumor fragment is 4mm × 4mm × 4mm.

In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue in each fragment. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue in each fragment. In some embodiments, the tumor fragmentation step is an in vitro or ex vivo method.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing action using a scalpel. In some embodiments, the tumor-indigestible liquid (TIL) is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically destroyed again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically destroyed a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether further incubation at 37°C in 5% _CO2 for 30 minutes is performed. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, the cell suspension prior to initiating the first amplification step is referred to as the “primary cell population” or “freshly acquired” or “freshly isolated” cell population.

In some embodiments, the cells may optionally be frozen after sample separation (e.g., after obtaining a tumor sample and/or after obtaining a cell suspension from a tumor sample) and cryopreserved before proceeding to the amplification described in step B, which is further described in detail below and illustrated in Figure 8 (particularly, for example, Figure 8B).

1. TIL derived from hollow needles/small biopsies

In some implementations, TILs are initially obtained from patient tumor samples (“primary TILs”) obtained through core needle biopsy or similar procedures and then expanded into a larger population for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters.

In some embodiments, patient tumor samples may be obtained using methods known in the art, typically via mini-biopsy, core needle biopsy, needle biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples may be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples may also be liquid tumors, such as those obtained from hematologic malignancies. In some embodiments, the sample may be derived from multiple small tumor samples or biopsies. In some embodiments, the sample may comprise multiple tumor samples from a single tumor from the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three, or four tumors from the same patient. In some embodiments, the sample may comprise multiple tumor samples from multiple tumors from the same patient. The solid tumor may be lung and/or non-small cell lung cancer (NSCLC).

Generally, cell suspensions obtained from the tumor core or fragments are referred to as “primary cell populations” or “freshly acquired” or “freshly isolated” cell populations. In some embodiments, the freshly acquired TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

In some implementations, if the tumor is metastatic and the primary lesion has been effectively treated/removed in the past, it may be necessary to remove a metastatic lesion. In some implementations, when available, minimally invasive methods are used to remove skin lesions or lymph nodes in the neck or axillary region. In some implementations, skin lesions are removed or small biopsies are taken. In some implementations, lymph nodes are removed or small biopsies are taken. In some implementations, the tumor is melanoma. In some implementations, the small biopsy of melanoma includes a mole or a portion thereof.

In some embodiments, a small biopsy is a perforated biopsy. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin surrounding a suspected mole. In some embodiments, a perforated biopsy is obtained by pressing a round blade into the skin and removing a circular piece of skin. In some embodiments, a small biopsy is a perforated biopsy and removes a circular portion of the tumor.

In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed along with a small border of normal-looking skin.

In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy and only the most irregular portion of the mole or growth is collected. In some implementations, the small biopsy is an incisional biopsy, and the incisional biopsy is used when other techniques cannot be performed, such as when the suspected mole is very large.

In some implementations, the small biopsy is a lung biopsy. In some implementations, the small biopsy is obtained via bronchoscopy. Typically, bronchoscopy involves inserting a small instrument through the nose or mouth, down the throat, and into the bronchial passage under the patient's anesthesia, where the instrument is used to remove tissue. In some implementations, a transthoracic biopsy may be used when the tumor or growth cannot be reached via bronchoscopy. Typically, for a transthoracic biopsy, the patient is also under anesthesia and a needle is inserted directly through the skin to the suspected site to remove a small tissue sample. In some implementations, a transthoracic biopsy may require interventional radiology (e.g., using X-ray or CT scan-guided needle). In some implementations, the small biopsy is obtained via puncture biopsy. In some implementations, the small biopsy is obtained via endoscopic ultrasound (e.g., an endoscope with a light and placed in the esophagus via the mouth). In some implementations, the small biopsy is obtained surgically.

In some implementations, the small biopsy is a head and neck biopsy. In some implementations, the small biopsy is an incisional biopsy. In some implementations, the small biopsy is an incisional biopsy in which a small piece of tissue is removed from the area of abnormal appearance. In some implementations, if the abnormal area is easily accessible, a sample can be collected without hospitalization. In some implementations, if the tumor is deep inside the mouth or throat, the biopsy may need to be performed under general anesthesia in an operating room. In some implementations, the small biopsy is an excisional biopsy. In some implementations, the small biopsy is an excisional biopsy in which the entire area is removed. In some implementations, the small biopsy is a fine-needle aspiration (FNA). In some implementations, the small biopsy is a fine-needle aspiration (FNA) in which cells are extracted (aspirated) from the tumor or mass using a very fine needle attached to a syringe. In some implementations, the small biopsy is a perforated biopsy. In some implementations, the small biopsy is a perforated biopsy in which a suspicious area is removed using perforated forceps.

In some implementations, the small biopsy is a cervical biopsy. In some implementations, the small biopsy is obtained via colposcopy. Typically, the colposcopy method uses a light-assisted magnifying instrument (coloscope) attached to a binocular magnifying lens, which is then used to examine a small portion of the cervix. In some implementations, the small biopsy is a cervical conization/cone biopsy. In some implementations, a small biopsy is a cervical conization/cone biopsy, in which an outpatient procedure may be required to remove a larger piece of tissue from the cervix. In some implementations, in addition to aiding in diagnosis, a cone biopsy can also be used as initial treatment.

The term "solid tumor" refers to an abnormal mass of tissue that typically does not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The histological structure of a solid tumor includes interdependent tissue compartments, comprising the parenchyma (cancer cells) and supporting stromal cells dispersed within it, providing a supportive microenvironment.

In some embodiments, samples from the tumor are obtained in the form of fine-needle aspiration (FNA), core needle biopsy, or mini-biopsy (including, for example, perforated biopsy). In some embodiments, the sample is first placed in a G-REX-10. In some embodiments, when one or two core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-10. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-100. In some embodiments, when three, four, five, six, eight, nine, or ten or more core needle biopsy and/or mini-biopsy samples are present, the sample is first placed in a G-REX-500.

FNA can be obtained from skin tumors, including, for example, melanoma. In some implementations, the FNA is obtained from a skin tumor, such as one from a patient with metastatic melanoma. In some cases, the patient with melanoma has previously undergone surgical treatment.

FNA can be derived from lung tumors, including, for example, NSCLC. In some implementations, the FNA is derived from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the NSCLC patient has previously undergone surgical treatment.

The TILs described herein may be obtained from FNA samples. In some cases, FNA samples are obtained or separated from the patient using fine-gauge needles ranging from 18 to 25 gauge. Fine-gauge needles may be 18, 19, 20, 21, 22, 23, 24, or 25 gauge. In some embodiments, the FNA sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

In some cases, the TILs described herein are obtained from core needle biopsy samples. In some cases, the core needle biopsy sample is obtained or separated from the patient using a surgical or medical needle in the range of 11 to 16 gauge. The needle may be 11, 12, 13, 14, 15, or 16 gauge. In some embodiments, the core needle biopsy sample from the patient may contain at least 400,000 TILs, such as 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or more.

Generally speaking, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some implementations, the TIL is not derived from tumor digest. In some implementations, the solid tumor core is not fragmented.

In some embodiments, the tumor digest is derived from the tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically dissociated again for approximately 1 minute. After incubation at 37°C in 5% _CO2 for another 30 minutes, the tumor can be mechanically dissociated a third time for approximately 1 minute. In some embodiments, if large areas of tissue remain after the third mechanical dissociation, one or two additional mechanical dissociations to the sample are applied, regardless of whether it is incubated at 37°C in 5% _CO2 for another 30 minutes. In some implementations, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

In some implementations, obtaining the first TIL population involves a multifocal sampling method.

Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersing enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, type XIV protease (streptolysin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof.

In some embodiments, the dissociative enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a sterile buffer such as Hank's balance salt solution (HBSS).

In some cases, collagenase (e.g., animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, for example, about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 15 ... Z U/mL, approximately 200 PZ U/mL, approximately 210 PZ U/mL, approximately 220 PZ U/mL, approximately 230 PZ U/mL, approximately 240 PZ U/mL, approximately 250 PZ U/mL, approximately 260 PZ U/mL, approximately 270 PZ U/mL, approximately 280 PZ U/mL, approximately 289.2 PZ U/mL, approximately 300 PZ U/mL, approximately 350 PZ U/mL, or approximately 400 PZ U/mL.

In some implementations, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after rehydration, the neutral protease stock solution ranges from 100 DMC/mL to about 400 DMC/mL, for example, about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, for example, about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some implementations, the enzyme stock solution may be varied, thus requiring verification of the concentration of the lyophilized stock solution and corresponding modification of the final amount of enzyme added to the digestion mixture.

In some embodiments, the enzyme mixture comprises approximately 10.2 μl of neutral protease (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of DNase I (200 U/mL) in approximately 4.7 mL of sterile HBSS.

2. Pleural effusion T cells and TILs

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of the T cells or TILs used for expansion according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of containing TILs can be used. Such samples may be derived from primary or metastatic lung cancers, such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells derived from another organ (e.g., breast, ovary, colon, or prostate). In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is pleural effusion. Other biological samples may include other serous fluids containing TILs, including, for example, ascites fluid or pancreatic cyst fluid from the abdomen. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and peritoneal cavities in the same malignant tumor events, and in some embodiments, such fluids contain TILs. In some embodiments of the pleural fluid in the examples of the present invention, ascites fluid or other cyst fluid containing TILs can be used for the same method to obtain similar results.

In some embodiments, pleural fluid is taken directly from the patient in its untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., an EDTA or heparin tube) prior to the contact step. In some embodiments, the untreated pleural fluid is placed in a standard tube (Veridex) prior to the contact step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of live TILs. If retained in untreated pleural fluid, the number of live TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid/diluent. In other embodiments, the dilution is 1:9 pleural fluid/diluent. In other embodiments, the dilution is 1:8 pleural fluid/diluent. In other embodiments, the dilution is 1:5 pleural fluid/diluent. In other embodiments, the dilution is 1:2 pleural fluid/diluent. In other embodiments, the dilution is 1:1 pleural fluid/diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of live TILs, which can significantly decrease within 24-48 hours if retained in untreated pleural fluid, even at 4°C. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted. In some embodiments, pleural fluid samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after being taken from the patient and diluted at 4°C.

In other embodiments, the pleural fluid sample is concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred when it must be cryopreserved for transport to the laboratory performing the method or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging it after removal from the subject and resuspending the centrifuged fluid or precipitate in a buffer solution. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspendings before being cryopreserved for transport or subsequent analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in the contact step is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 μM. In other embodiments, the pore size may be 5 μM or larger, and in other embodiments, it may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained on the membrane may be flushed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in the contact step of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially dissolves enucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in cases where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent, or a lysis reagent and a quenching agent, or a lysis reagent, a quenching agent, and a fixative agent. Suitable lysis systems are commercially available and include the BD Pharm Lyse ^™ system (Becton Dickenson). Other lysis systems include the Versalyse ^™ system, the FACSlyse ^™ system (Becton Dickenson), the Immunoprep ^™ system, or the Erythrolyse II system (Beckman Coulter) or an ammonium chloride system. In some embodiments, the lysis reagent may vary depending on primary requirements such as efficient lysis of red blood cells and conservation of TILs and phenotypic characteristics of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system suitable for the methods described herein may include a second reagent, such as a second reagent that quenches or delays the action of the dissolving reagent during the remaining steps of the method, for example, Stabilize ^™ reagent (Beckman Coulter). Conventional fixatives may also be used, depending on the choice of dissolving reagent or preferred implementation of the method.

In some implementations, pleural fluid samples, as described above, are cryopreserved at approximately -140°C and then subjected to further processing and/or amplification as provided herein.

3. Methods for expanding peripheral blood lymphocytes (PBL) from peripheral blood.

PBL Method 1. In some embodiments of the invention, PBL is amplification using the methods described herein. In some embodiments of the invention, the method includes obtaining a PBMC sample from whole blood. In some embodiments, the method includes enriching T cells by using negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC. In some embodiments, the method includes enriching T cells by using magnetic bead-based negative selection of a non-CD19+ fraction to isolate pure T cells from the PBMC.

In some embodiments of the present invention, PBL method 1 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. T cells are isolated using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

PBL Method 2. In some embodiments of the invention, PBL is amplified using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells from the PBMCs are enriched by incubating the PBMCs at 37°C for at least three hours and then separating non-adhesive cells.

In some embodiments of the present invention, PBL method 2 is performed as follows: On day 0, the frozen PMBC sample is thawed, and PBMC cells are seeded at 6 million cells per well in 6-well plates of CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, the non-adherent cells (which are PBL) are removed and their number is counted.

PBL Method 3. In some embodiments of the invention, PBL is amplified using PBL Method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection and T cells are selected using a non-CD19+ fraction of the PBMC sample using negative selection.

In some embodiments of the present invention, PBL method 3 is performed as follows: On day 0, frozen PBMCs derived from peripheral blood are thawed and their number is counted. CD19+ B cells are sorted using a CD19 multi-sorting human kit (Miltenyi Biotec). In the non-CD19+ cell fraction, T cells are purified using a human pan-T cell isolation kit and an LS column (Miltenyi Biotec).

In some embodiments, PBMCs are isolated from whole blood samples. In some embodiments, PBMC samples are used as starting material for amplifying PBL. In some embodiments, the samples are cryopreserved prior to the amplification process. In other embodiments, fresh samples are used as starting material for amplifying PBL. In some embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human pan-T cell isolation kit and an LS column. In some embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art (e.g., CD19 negative selection).

In some embodiments of the invention, the PBMC sample is incubated for a period of time at a temperature required for effective identification of non-adhesive cells. In some embodiments of the invention, the incubation time is approximately 3 hours. In some embodiments of the invention, the temperature is approximately 37°C. The non-adhesive cells are then amplified using the process described above.

In some embodiments, PBMC samples are from subjects or patients who have optionally pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, tumor samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor. In some embodiments, PBMC samples are from subjects or patients who have pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or longer. In other embodiments, PBMCs are from patients currently receiving an ITK inhibitor regimen (e.g., ibrutinib).

In some implementations, PBMC samples are derived from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor and who are difficult to treat with a kinase inhibitor or an ITK inhibitor (e.g., ibrutinib).

In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment. In some embodiments, PBMC samples are from subjects or patients who have been pretreated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving kinase inhibitor or ITK inhibitor treatment and have not been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or longer. In other embodiments, PBMCs are derived from patients who have previously been exposed to ITK inhibitors but have not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In some embodiments of the invention, on day 0, cells are selected for CD19+ and sorted accordingly. In some embodiments of the invention, antibody-bound beads are used for selection. In some embodiments of the invention, purified T cells are isolated from PBMCs on day 0.

In some embodiments of the present invention, for patients who have not been pretreated with ibrutinib or other ITK inhibitors, 10-15 mL of erythrocyte sedimentation rate (ESR) amber layer will produce approximately 5 × ^10⁹ PBMCs, which in turn will produce approximately 5.5 × ^10⁷ PBLs.

In some embodiments of the invention, for patients pretreated with ibrutinib or other ITK inhibitors, the amplification process will generate approximately 20 × ^10⁹ PBLs. In some embodiments of the invention, 40.3 × ^10⁶ PBMCs will generate approximately 4.7 × ^10⁵ PBLs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or obtained from any other method known in the art for obtaining PBMCs.

In some embodiments, the PBL is prepared using the method described in U.S. Patent Application Publication No. US 2020/0347350A1, the disclosure of which is incorporated herein by reference.

4. Methods for expanding bone marrow infiltrating lymphocytes (MILs) derived from bone marrow-derived PBMCs

MIL Method 3. In some embodiments of the invention, the method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+/CD33+/CD20+/CD14+ cells, and non-CD3+/CD33+/CD20+/CD14+ cell fractions are sonicated and a portion of the sonicated cell fractions are added back to the selected cell fractions.

In some embodiments of the present invention, MIL method 3 is performed as follows: On day 0, the frozen PBMC sample is thawed and the number of PBMCs is counted. Cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using an S3e cell sorter (Bio-Rad). Cells are sorted into two fractions: immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and AML embryonic cell fraction (non-CD3+CD33+CD20+CD14+).

In some embodiments of the invention, PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow via apheresis, aspiration, biopsy, or other similar methods known in the art. In some embodiments, PBMCs are fresh. In other embodiments, PBMCs are cryopreserved.

In some embodiments of the invention, the MIL is expanded from 10-50 mL of bone marrow aspirate. In some embodiments of the invention, 10 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 20 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 30 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 40 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 50 mL of bone marrow aspirate is obtained from the patient.

In some embodiments of the invention, the number of PBMCs produced from about 10-50 mL of bone marrow aspirate is about 5 × ^10⁷ to about 10 × ^10⁷ PBMCs. In other embodiments, the number of PBMCs produced is about 7 × ^10⁷ PBMCs.

In some embodiments of the invention, approximately 5 × ^10⁷ to approximately 10 × ^10⁷ PBMCs produce approximately 0.5 × ^10⁶ to approximately 1.5 × ^10⁶ MILs. In some embodiments of the invention, approximately 1 × ^10⁶ MILs are produced.

In some embodiments of the invention, 12 × ^10⁶ PBMCs derived from bone marrow aspirate yield approximately 1.4 × ^10⁵ MILs.

In any of the foregoing embodiments, PBMCs may be derived from whole blood samples, bone marrow, obtained by apheresis, derived from the erythrocyte sedimentation rate (ESR) layer, or from any other method known in the art for obtaining PBMCs.

In some embodiments, the MIL is prepared using the method described in U.S. Patent Application Publication No. US2020/0347350A1, the disclosure of which is incorporated herein by reference.

Step B: Initiating the first amplification

In some embodiments, the method of the present invention provides younger TILs that may offer additional therapeutic benefits compared to older TILs (i.e., TILs that have undergone further replication in multiple rounds prior to administration to the subject/patient). Characteristics of younger TILs have been described in the literature, for example, Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32, 415-423; Robbins et al., J. Immunother. 2004, 173, 7125-7130; Shen et al., J. Immunother. 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother. 2008, 31, 742-751, each of which is incorporated herein by reference.

Following the segmentation or digestion of tumor fragments and/or tumor fragments as described in step A of, for example, Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) under conditions favorable to TIL growth but unfavorable to tumor and other cell growth. In some embodiments, IL-2, OKT-3, and feeder cells are added together with the tumor digest and/or tumor fragments at the start of culture (e.g., on day 0). In some embodiments, the tumor digest and/or tumor fragments are incubated in containers at a concentration of up to 60 fragments per container and with 6000 IU/mL IL-2. In some embodiments, culturing this primary cell population for several days, typically 1 to 8 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, culturing this primary cell population for several days, typically 1 to 7 days, yields a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 1 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 8 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, initiating the first expansion occurs over a period of 5 to 7 days, yielding a primary TIL population of approximately 1 × ^10⁸ main TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 6 to 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 to 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 7 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells. In some embodiments, this initial expansion occurs over a period of approximately 8 days, resulting in a host TIL population of approximately 1 × ^10⁸ host TIL cells.

In some implementations, TIL amplification may be performed using the steps described in step B of Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), which may include a process called pre-REP or initiating REP and which begins on day 0 and/or from the start of culture containing feeder cells), followed by a rapid second amplification (step D, including a process called the Rapid Amplification Protocol (REP) step) as described in step D below and here, followed by optional cryopreservation, and then a second step D as described below and here (including a process called the restimulation REP step). TILs obtained from this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some implementations, the tumor fragments are between approximately 1 ^mm³ and 10 ^mm³ .

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.

In some embodiments, there are fewer or equal to 240 tumor fragments. In some embodiments, fewer or equal to 240 tumor fragments are placed in fewer or equal to 4 containers. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, fewer or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each container contains fewer or equal to 500 mL of culture medium. In some embodiments, the culture medium contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the culture medium contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains OKT-3. In some embodiments, the culture medium contains 30 ng/mL OKT-3 per container. In some embodiments, the containers are GREX100 MCS culture flasks. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells ^per container.

After preparing the tumor fragment, the resulting cells (i.e., fragments of the primary cell population) are cultured in a medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions favorable to TIL growth but unfavorable to tumor and other cell growth, allowing TIL initiation and accelerated growth from day 0. In some embodiments, the tumor digest and/or tumor fragment are incubated with 6000 IU/mL IL-2, antigen-presenting feeder cells, and OKT-3. This primary cell population is cultured for several days, typically 1 to 8 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initial expansion period contains IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for several days, typically 1 to 7 days, yielding a primary TIL population typically of about 1 × ^10⁸ host TIL cells. In some embodiments, the growth medium during the initiation of the first expansion comprises IL-2 or a variant thereof, along with antigen-presenting feeder cells and OKT-3. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20-30 ^{× 10⁶} IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 × 10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the initial amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the initial amplification medium contains about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the initial expansion medium contains about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some embodiments, the initial expansion cell culture medium also contains IL-2. In some embodiments, the initial expansion cell culture medium contains about 3,000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains approximately 1000 IU/mL, approximately 1500 IU/mL, approximately 2000 IU/mL, approximately 2500 IU/mL, approximately 3000 IU/mL, approximately 3500 IU/mL, approximately 4000 IU/mL, approximately 4500 IU/mL, approximately 5000 IU/mL, approximately 5500 IU/mL, approximately 6000 IU/mL, approximately 6500 IU/mL, approximately 7000 IU/mL, approximately 7500 IU/mL, or approximately 8000 IU/mL IL-2. In some implementations, the initial cell expansion culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or about 8000 IU/mL IL-2.

In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the initial expansion medium contains approximately 200 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the initial expansion cell culture medium also contains IL-15. In some embodiments, the initial expansion cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the initial amplification medium contains about 2 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the initial amplification cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the initial amplification cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the initial cell culture medium for expansion contains OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 30 ng/mL of OKT-3 antibody. In some embodiments, the initial cell culture medium for expansion contains approximately 0.1 ng/mL, approximately 0.5 ng/mL, approximately 1 ng/mL, approximately 2.5 ng/mL, approximately 5 ng/mL, approximately 7.5 ng/mL, approximately 10 ng/mL, approximately 15 ng/mL, approximately 20 ng/mL, approximately 25 ng/mL, approximately 30 ng/mL, approximately 35 ng/mL, approximately 40 ng/mL, approximately 50 ng/mL, approximately 60 ng/mL, approximately 70 ng/mL, approximately 80 ng/mL, approximately 90 ng/mL, approximately 100 ng/mL, approximately 200 ng/mL, approximately 500 ng/mL, and approximately 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is moromboclovir. See, for example, Table 1.

In some embodiments, the initial amplification cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the initiating first expansion cell culture medium also contains IL-2 at an initial concentration of about 6000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein said one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the initiation first expansion medium is referred to as "CM" (an abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM HEpes, and 10 mg/mL gentamicin. In some embodiments, CM is CM1 as described in the examples. In some embodiments, the initiation of the first expansion is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the initiation first expansion medium or initial cell culture medium or first cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (also referred to herein as feeder cells).

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific). In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some implementations, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisherScientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

In some embodiments, the specific culture medium described by reference to International PCT Publication WO/1998/030679, which is incorporated herein by reference, can be used in this invention. In that disclosure, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4+, Br, T, ^{Mn2 +} , P, Si4+, V5 ⁺ , Mo6 ⁺ , ^{Ni2 +} , Rb+, Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

Table 4: Concentration of Non-Trace Element Components

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) can be used in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 4 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 5 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG1 or FIG36 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 7 to 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 8 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 1 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 2 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 3 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 4 to 7 days, as discussed in the embodiments and figures. In some embodiments, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8B and/or FIG8C), which may include those processes sometimes referred to as pre-REP or REP initiation) takes 5 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) takes 6 to 7 days, as discussed in the embodiments and figures. In some implementations, the initiation of the first amplification process (including, for example, those processes described in step B of FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), which may include those processes sometimes referred to as pre-REP or initiation of REP) is 7 days, as discussed in the embodiments and figures.

In some embodiments, the initiation of first TIL amplification may be performed 1 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 1 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 2 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 3 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 4 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 5 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 6 to 7 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 7 to 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some embodiments, the initiation of first TIL amplification may be performed 8 days after fragmentation occurs and/or after the start of the first initiation amplification step. In some implementations, the initiation of the first TIL amplification can be performed 7 days after fragmentation occurs and/or after the start of the first initiation amplification step.

In some implementations, the initial amplification of the TIL can take place for 1, 2, 3, 4, 5, 6, 7, or 8 days. In some implementations, the first TIL amplification can take place for 1 to 8 days. In some implementations, the first TIL amplification can take place for 1 to 7 days. In some implementations, the first TIL amplification can take place for 2 to 8 days. In some implementations, the first TIL amplification can take place for 2 to 7 days. In some implementations, the first TIL amplification can take place for 3 to 8 days. In some implementations, the first TIL amplification can take place for 3 to 7 days. In some implementations, the first TIL amplification can take place for 4 to 8 days. In some implementations, the first TIL amplification can take place for 4 to 7 days. In some implementations, the first TIL amplification can take place for 5 to 8 days. In some implementations, the first TIL amplification can take place for 5 to 7 days. In some implementations, the first TIL amplification can take place for 6 to 8 days. In some implementations, the first TIL amplification can take place for 6 to 7 days. In some implementations, the first TIL amplification can take place for 7 to 8 days. In some implementations, the first TIL amplification can take 8 days. In some implementations, the first TIL amplification can take 7 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, during the initiation of the first amplification, IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included, for example, during step B of the process described herein, according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as the combination during the initiation of the first amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step B.

In some embodiments, step B of initiating the first amplification (e.g., according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

1. Feeder cells and antigen-presenting cells

In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added during the initiation of the first amplification. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 4–7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 5-7 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during days 6–8 of the initiation first amplification period. In some implementations, the initiation first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 6-7 of the initiation first amplification period. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 or 8 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 7 of the initiation of the first amplification. In some implementations, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as pre-REP or initiation REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification, but is added at any time during day 8 of the initiation of the first amplification.

In some embodiments, the initiation of the first amplification procedure described herein (e.g., including amplifications such as those described in step B of Figure 8 (especially, for example, Figure 8B) and those amplifications referred to as pre-REP or initiation REP) requires feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL amplification and during the initiation of the first amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, 2.5 × ^10⁸ feeder cells are used during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per container during the initiation of the first amplification. In some embodiments, 2.5 × ^{10⁸ feeder cells are used per GREX-10} during the initiation of the first amplification. In some embodiments, 2.5 × ^10⁸ feeder cells are used per GREX-100 during the initiation of the first amplification.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 has not increased compared to the initial number of viable cells introduced into the culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the first expansion, the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the initiation of the first expansion described herein requires approximately 2.5 × ^10⁸ feeder cells. In other embodiments, the number of feeder cells required to initiate the first expansion is one-quarter, one-third, five-twelfths, or one-half of the number of feeder cells used for rapid second expansion.

In some embodiments, the culture medium for initiating the first expansion contains IL-2. In some embodiments, the culture medium for initiating the first expansion contains 6000 IU/mL IL-2. In some embodiments, the culture medium for initiating the first expansion contains antigen-presenting feeder cells. In some embodiments, the culture medium for initiating the first expansion contains 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium for initiating the first expansion contains OKT-3. In some embodiments, the culture medium contains 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells. In some embodiments, the culture medium comprises 500 mL of culture medium and ¹⁵ μg OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium comprises 500 mL of culture medium, 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg OKT-3 per container for every 2.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the initiation of the first expansion procedure described herein requires an excess of feeder cells beyond the TIL during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs to initiate the first expansion.

2. Cytokines and other additives

The amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2), as known in the art.

Alternatively, it is also possible to initiate the first expansion of TILs using cytokines in combination with two or more of IL-2, IL-15, and IL-21 as described in U.S. Patent Application Publication No. US2017/0107490 A1, the disclosure of which is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having a particular use in many embodiments. The use of combinations of cytokines is particularly beneficial for the generation of lymphocytes, and especially T cells as described therein. See, for example, Table 2.

In some embodiments, step B may further include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may further include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Additionally, additives may be used in the culture medium during step B, such as peroxisome proliferator-activated receptor gamma coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: Initiating the transition from the first amplification to the rapid second amplification

In some cases, a population of primary TILs obtained from an initial first amplification (which may include amplification sometimes referred to as pre-REP), including, for example, a population of TILs obtained from step B as indicated in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), may undergo a rapid second amplification (which may include amplification sometimes referred to as a rapid amplification protocol (REP)) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs are intended for therapeutic use, an amplified TIL population from an initial first amplification or an amplified TIL population from a rapid second amplification may be genetically modified for appropriate treatment before the amplification step or after the initial first amplification and before the rapid second amplification.

In some implementations, the TIL obtained from step B as indicated in the initiation of the first amplification (e.g., Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is stored until the phenotype is determined for selection. In some embodiments, the TIL obtained from the initiation of the first amplification (e.g., step B as indicated in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is not stored and is directly subjected to rapid second amplification. In some embodiments, the TIL obtained from the initiation of the first amplification is not cryopreserved after the initiation of the first amplification and before rapid second amplification. In some embodiments, the transition from the initiation of the first amplification to the second amplification occurs approximately 2, 3, 4, 5, 6, 7, or 8 days after tumor fragmentation occurs and/or after the initiation of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs approximately 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs approximately 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 6 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 to 8 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 7 days after fragmentation and/or after the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs approximately 8 days after fragmentation and/or after the start of the first initiation amplification step.

In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1, 2, 3, 4, 5, 6, 7, or 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 1 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 2 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to second amplification occurs 3 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs 3 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 4 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 5 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition from initiating the first amplification to rapid second amplification occurs 6 to 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 6 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 to 8 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 7 days after fragmentation and/or the start of the first initiation amplification step. In some embodiments, the transition occurs 8 days after fragmentation and/or the start of the first initiation amplification step.

In some embodiments, TILs are not stored after the initial first amplification and before the rapid second amplification, and the TILs undergo rapid second amplification directly (e.g., in some embodiments, they are not stored during the transition from step B to step D as shown in Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TILs from which the first amplification was initiated (the second TIL population) undergo rapid second amplification directly without a transition period.

In some embodiments, the transition from initiating the first amplification to the rapid second amplification (e.g., step C according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed-system TIL amplification as described herein is performed. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a GREX-10 or GREX-100. In some embodiments, the closed-system bioreactor is a single bioreactor. In some embodiments, the transition from initiating the first amplification to the rapid second amplification involves a longitudinal scaling up of the container size. In some embodiments, the initiation of the first amplification is carried out in a smaller container compared to the rapid second amplification. In some implementations, the initial amplification is performed in a GREX-100 and the rapid second amplification is performed in a GREX-500.

In some embodiments, the TIL obtained from the first amplification (e.g., step B shown in Figures 36A-D or 8I-P) is converted to the activation or gene editing step. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 12 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the activation or gene editing step occurs approximately 3 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 3 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 5 days after fragmentation. In some embodiments, the transition occurs approximately 3 to 4 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 13 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 4 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 7 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 4 to 5 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 8 days after fragmentation. In some embodiments, the transition occurs approximately 5 to 7 days after fragmentation. In some embodiments, the transition from the first amplification to activation or gene editing occurs approximately 5 to 6 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 14 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 13 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 12 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 11 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 10 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 9 days after fragmentation. In some embodiments, the transition occurs approximately 6 to 8 days after fragmentation. In some implementations, the transition from the first amplification to activation or gene editing steps occurs approximately 6 to 7 days after fragmentation.

In some implementations, the transition from the first amplification to the activation or gene editing step occurs in a closed system, as described herein.

In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and/or anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing anti-CD3 and anti-CD38 beads. In some embodiments, the activation step includes culturing TILs for approximately 1-7 days in a medium containing OKT3. In some embodiments, the activation step is performed for approximately 1-7 days. In some implementations, the activation step takes approximately 1-7 days, approximately 1-6 days, approximately 2-6 days, approximately 3-6 days, approximately 4-6 days, approximately 5-6 days, approximately 1-5 days, approximately 2-5 days, approximately 3-5 days, approximately 4-5 days, approximately 1-4 days, approximately 2-4 days, approximately 3-4 days, approximately 1-3 days, approximately 2-3 days, or approximately 1-2 days. In some implementations, the activation step takes approximately 1 day. In some implementations, the activation step takes approximately 2 days. In some implementations, the activation step takes approximately 3 days. In some implementations, the activation step takes approximately 4 days. In some implementations, the activation step takes approximately 5 days. In some implementations, the activation step takes approximately 6 days. In some implementations, the activation step takes approximately 7 days.

In view of this specification, any suitable anti-CD3/anti-CD38 beads known in the art may be used. Suitable anti-CD3/anti-CD38 beads include, but are not limited to, commercially available products, including, but not limited to, Dynabeads ^™ Human T-Activator CD3/CD28 (available from Invitrogen), ImmunoCult ^™ Human CD3/CD28 T-Cell Activator (available from StemCell Technologies), and T Cell TransAct ^™ (available from Miltenyi Biotec) for T-cell expansion and activation.

In some implementations, the activation step is optional. In some implementations, the activation step is optional if the first amplification includes OKT-3.

In some implementations, the TIL obtained from the first amplification (e.g., step B as shown in Figures 36C-D) or from the activation step (e.g., step C as shown in Figures 36A-B) is converted into a gene editing step.

In some embodiments, the gene editing step includes aseptic electroporation of the TIL population. In some embodiments, the aseptic electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CTLA-4 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating LAG-3 expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CISH expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating CBL-B expression. According to some embodiments, the gene editor further comprises a TALE nuclease system for downregulating TIGIT expression. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a CTLA-4 gene knockout TIL. According to some embodiments, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of LAG-3 expression and downregulation of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CISH expression and downregulation of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulation of CBL-B expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/CBL-B double gene knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/TIGIT double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/LAG-3 double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CISH double gene knockout TILs. According to some implementation schemes, the resulting TILs are CTLA-4/CBL-B double gene knockout TILs. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CBL-B/TIGIT double gene knockout TIL.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or Figures 8I-P) is converted to a second electroporation step. In some embodiments, a resting step exists between the first and second electroporation steps. In some embodiments, the resting step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, or approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 1.5 days, approximately 2 days, approximately 2.5 days, or approximately 3 days. In some embodiments, a second electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates PD-1 expression. According to some embodiments, the TALE nuclease system downregulates CTLA-4. According to some embodiments, the TALE nuclease system downregulates CISH expression. According to some embodiments, the TALE nuclease system downregulates LAG-3 expression. According to some implementation schemes, the TALE nuclease system downregulates TIGIT expression. According to some implementation schemes, the TALE nuclease system downregulates CBL-B expression. According to some implementation schemes, the resulting TIL is a PD-1 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CTLA-4 gene knockout TIL. According to some implementation schemes, the resulting TIL is a LAG-3 gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulation of PD-1 expression and downregulation of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulation of CTLA-4 expression and downregulation of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of LAG-3 and one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CISH and one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some implementation schemes, the resulting TILs exhibit downregulated expression of CBL-B and one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some implementation schemes, the resulting TILs exhibit downregulated expression of TIGIT and one or more of CTLA-4, LAG-3, CISH, CBL-B, and PD-1. According to some implementation schemes, the resulting TILs are PD-1/CTLA-4 double knockout TILs. According to some implementation schemes, the resulting TILs are PD-1/LAG-3 double knockout TILs. According to some implementation schemes, the obtained TIL is a PD-1/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the resulting TIL is a CBL-B/TIGIT double gene knockout TIL.

In some implementations, the gene editing process also includes a quiescent step. According to some implementations, the quiescent step involves incubating a fourth TIL population at approximately 30-40°C and approximately 5% _CO2 . According to some implementations, the quiescent step is performed at approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, approximately 33.5°C, approximately 34°C, approximately 34.5°C, approximately 35°C, approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, approximately 37.5°C, approximately 38°C, approximately 38.5°C, approximately 39°C, approximately 39.5°C, and approximately 40°C. According to some embodiments, the resting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the resting step includes incubating a third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for approximately one hour, followed by incubation at approximately 30°C for approximately 15 hours to approximately 23 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 16 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 17 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 18 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 19 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 20 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 21 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 22 hours. According to some embodiments, the quiescent step includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 23 hours.

In some embodiments, the TIL obtained from the gene editing step or resting step (e.g., step C shown in Figures 36A-D or 8I-P) is converted to the second amplification step (e.g., step D shown in Figures 36A-D or 8I-P). In some embodiments, the transition from the gene editing step or resting step to the second amplification step occurs approximately 0.5 days, 1 day, 2 days, 3 days, or 4 days after the start of the gene editing step or resting step.

In some implementations, the transition from the gene editing step or quiescent step to the second amplification step occurs in a closed system, as described herein.

In some implementations, the TIL is not stored after the gene editing step or the resting step and before the second amplification, and the TIL is directly used for the second amplification (e.g., in some implementations, it is not stored during the transition from step C to step D, as shown in Figures 36A-D or Figures 8I-P).

In some embodiments, the transition from the first amplification to the second amplification, for example according to step C of Figure 36, is carried out in a closed-system bioreactor. In some embodiments, the closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed-system bioreactor is a single bioreactor.

Step D: Rapid Second Amplification

In some implementations, the TIL cell population is further expanded in number after the collection and initiation of the first expansion (steps A and B) and the transition referred to as step C, as indicated in Figures 8 (in particular, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). This further amplification, referred to herein as rapid second amplification or rapid amplification, may include an amplification process commonly referred to in the art as a rapid amplification procedure (rapid amplification protocol or REP); and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Rapid second amplification is typically performed in a ventilated container using a culture medium containing multiple components, including feeder cells, cytokine sources, and anti-CD3 antibodies. In some embodiments, the TIL is transferred to a larger volume container 1, 2, 3, or 4 days after the initiation of rapid second amplification (i.e., on days 8, 9, 10, or 11 of the overall Gen 3 process).

In some embodiments, the rapid second amplification of TIL (which may include amplification sometimes referred to as REP; and the process indicated in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 2 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 3 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 4 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 5 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur from approximately 6 to approximately 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 9 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 8 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 9 to 10 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 1 day after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 2 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 3 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 4 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 5 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 6 days after the initiation of rapid second amplification. In some embodiments, the second TIL amplification may occur approximately 7 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 8 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 9 days after the initiation of rapid second amplification. In some implementations, the second TIL amplification may be performed approximately 10 days after the initiation of rapid second amplification.

In some embodiments, the rapid second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, amplification referred to as REP; and the process indicated in step D of FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, TIL is amplified in the rapid second amplification in the presence of IL-2, OKT-3, and feeder cells, wherein feeder cells are added to a final concentration that is two, 2.4, 2.5, 3, 3.5, or 4 times the feeder cell concentration present in the initiating first amplification. For example, TILs can be rapidly expanded by stimulation with nonspecific T cell receptors in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulants may include, for example, anti-CD3 antibodies, such as approximately 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Laritan, NJ or Miltenyi Biotech, Auburn, California), or UHCT-1 (available from BioLegend, San Diego, California). TILs can be amplified during a second amplification period to induce further in vitro stimulation of TILs by including one or more cancer antigens (including their antigenic portions, such as epitopes), said antigens optionally expressed from a vector in the presence of T cell growth factors (e.g., 300 IU/mL IL-2 or IL-15), said vectors being, for example, human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or their antigenic portions. TILs can also be rapidly amplified by pulsed stimulation with the same cancer antigens on antigen-presenting cells expressing HLA-A2. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In some embodiments, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng/mL to 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL to 60 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3. In some embodiments, the cell culture medium comprises about 60 ng/mL of OKT-3. In some embodiments, the OKT-3 antibody is moromboclovir.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium in the rapid second amplification contains 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium contains 500 mL of culture medium and 6000 IU/mL IL-2, 30 μg of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells per container.

In some embodiments, the culture medium in the rapid second amplification contains IL-2. In some embodiments, the culture medium contains 6000 IU/mL IL-2. In some embodiments, the culture medium in the rapid second amplification contains antigen-presenting feeder cells. In some embodiments, the culture medium contains 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells per container. In some embodiments, the culture medium in the rapid second amplification contains OKT-3. In some embodiments, the culture medium in the rapid second amplification contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS culture flask. In some embodiments, the culture medium in the rapid second amplification contains 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells. In some implementations, the culture medium in the rapid second amplification comprises 500 mL of culture medium per container and 6000 IU/mL IL-2, 30 μg OKT-3, and 5 × ^10⁸ to 7.5 × ^10⁸ antigen-presenting feeder cells.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and said 4-1BB agonist is selected from the group consisting of urilumab, utumumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and said one or more TNFRSF agonists contain a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification period. In some embodiments, during the second amplification period, including, for example, during step D of the process described herein according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P), IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification period. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) and as described herein in step D.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

In some embodiments, the second amplification medium contains about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second amplification medium contains approximately 200 IU/mL IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15. In some embodiments, the cell culture medium also contains IL-15. In some embodiments, the cell culture medium contains approximately 180 IU/mL IL-15.

In some embodiments, the second amplification medium contains about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second amplification medium contains about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second amplification medium contains about 2 IU/mL IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium also contains IL-21. In some embodiments, the cell culture medium contains about 1 IU/mL IL-21.

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen-presenting cells in rapid expansion and/or secondary expansion is approximately 1:10, approximately 1:15, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:75, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to PBMCs in rapid expansion and/or secondary expansion is between 1:50 and 1:300. In some implementations, the ratio of TIL to PBMC in rapid amplification and/or secondary amplification is between 1:100 and 1:200.

In some implementations, the REP and/or rapid second amplification are performed in a culture flask, wherein the host TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 ng/mL OKT3 anti-CD3 antibody, and 6000 IU/mL IL-2 in 150 mL of culture medium, wherein the feeder cell concentration is at least 1.1-fold (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.8X, 2X, 2.1X, 2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, or 4.0X. The culture medium is replaced (usually by removing 2/3 of the used medium and replacing it with an equal volume of fresh medium) until the cells are transferred to a replacement growth chamber. The replacement growth chamber consists of G-REX culture flasks and aeration containers, as discussed in more detail below.

In some embodiments, the rapid second amplification (which may include a process referred to as the REP process) takes 7 to 9 days, as discussed in the examples and figures. In some embodiments, the second amplification takes 7 days. In some embodiments, the second amplification takes 8 days. In some embodiments, the second amplification takes 9 days.

In some embodiments, the second amplification (which may include an amplification referred to as REP, and the amplification mentioned in step D of Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) can be performed in a 500 mL volumetric aerated culture flask (G-REX-100, available from Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA) with a 100 cm aerated silica bottom, 5 × ^10⁶ or 10 × ^10⁶ TILs can be taken together with PBMCs in 400 mL of supplemented 5% human AB serum at 3000 IU/mL Cells were cultured in a 50/50 medium containing IL-2 and 30 ng/mL anti-CD3 (OKT3). G-REX-100 culture flasks were incubated at 37°C in 5% _CO2 . On day 5, 250 mL of the supernatant was removed and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL precipitate was resuspended in 150 mL of fresh medium containing 5% human AB serum and 6000 IU/mL IL-2 and added back to the original GREX-100 culture flask. As TILs were continuously amplified in GREX-100 culture flasks, they could be transferred to larger culture flasks, such as GREX-500, on day 10 or 11. Cells could be collected on day 14. Cells could be collected on day 15. Cells could be collected on day 16. In some embodiments, the medium was replaced until the cells were transferred to a replacement growth chamber. In some embodiments, two-thirds of the culture medium is replaced by aspirating the used medium and replacing it with an equal volume of fresh medium. In some embodiments, alternative growth chambers include GREX culture flasks and aerated containers, as discussed more fully below. In some embodiments, the process employs different centrifugation speeds (400g, 300g, 200g, for 5 minutes) and different numbers of replicates.

In some embodiments, the culture medium used in the amplification process disclosed herein is a serum-free or defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or a serum substitute. In some embodiments, the serum-free or defined culture medium is used to prevent and/or reduce some of the experimental variations caused by batch-to-batch variations in serum-containing culture media.

In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and/or serum substitute. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CTS ^™ AIM-VSFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some implementations, the serum supplement or serum substitute includes (but is not limited to) one or more of the following: CTS ^™ OpTmizer T-cell expansion serum supplement, CTS ^™ immune cell serum substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , Al3 ⁺ , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , Cr3 ⁺ , Ge4 ⁺ , Se4 ⁺ , Br, T, ^Mn2+ , P, Si4 ⁺ , V5 ⁺ , Mo6 ⁺ , Ni2 ⁺ , Rb ⁺ , Sn2 ⁺ , and Zr4 ⁺ . In some embodiments, the culture medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some implementations, the CTS ^™ OpTmizer ^™ T cell immune cell serum substitute is used with conventional growth media, including (but not limited to) CTS ^™ OpTmizer ^™ T cell expansion basal medium, CTS ^™ OpTmizer ^™ T cell expansion SFM, CTS ^™ AIM-V medium, CST ^™ AIM-V SFM, LymphoONE ^™ T cell expansion Xeno-free medium, Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium, and Iskoff modified Dürbeco medium.

In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined culture medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined culture medium.

In some embodiments, the serum-free or defined culture medium is CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol.

In some embodiments, the culture medium is identified as CTS ^™ OpTmizer ^™ T-cell expansion SFM (ThermoFisher Scientific). Any CTS ^™ OpTmizer ^™ formulation can be used in this invention. CTS ^™ OpTmizer ^™ T-cell expansion SFM is a combination of 1 L of CTS ^™ OpTmizer ^™ T-cell expansion basal culture medium and 26 mL of CTS ^™ OpTmizer ^™ T-cell expansion supplement, which are mixed together prior to use. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and also contains approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 3000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains approximately 1000 IU/mL to approximately 6000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS ^™ OpTmizer ^™ T cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 3000 IU/mL IL-2. In some implementations, CTS ^™ OpTmizer ^™ T-cell expansion SFM is supplemented with approximately 3% CTS ^™ immune cell serum substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and also contains approximately 6000 IU/mL IL-2.

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM (i.e., ...). In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with glutamine at a concentration of about 2 mM (i.e., ...).

In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at concentrations of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free culture medium or the defined culture medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

In some embodiments, the specific culture media described in International Patent Application Publication No. WO 1998/030679 and U.S. Patent Application Publication No. US2002/0076747A1 (which are incorporated herein by reference) are applicable to the present invention. In the disclosures, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprise a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more of the following: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the specific culture medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the culture medium is defined to contain albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the culture medium is defined as containing albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and compounds containing trace element fractions of Ag ⁺ , ^{Al3 +} , Ba2 ⁺ , Cd2 ⁺ , Co2 ⁺ , ^{Cr3 +} , Ge4 ⁺ , Se4+, Br, T, ^Mn2+ , P, ^Si4+ , V5 ⁺ , Mo6 ⁺ , ^{Ni2 +, Rb+} , Sn2 ⁺ , and Zr4 ⁺ . In some implementations, the basal cell culture medium is selected from the group consisting of: Dürbeco modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow minimum essential medium (G-MEM), RPMI growth medium and Iskov modified Dürbeco medium.

In some embodiments, the concentrations of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, and L-tryptophan in the culture medium are determined to be in the range of about 5-200 mg/L, about 5-250 mg/L, about 1-1000 mg/L, about 1-45 mg/L, about 1-250 mg/L, about 10-500 mg/L, and about [missing information - likely a specific concentration range]. The concentrations of L-tyrosine (approximately 3-175 mg/L), L-valine (approximately 5-500 mg/L), thiamine (approximately 1-20 mg/L), reduced glutathione (approximately 1-20 mg/L), L-ascorbic acid-2-phosphate (approximately 1-200 mg/L), iron-saturated transferrin (approximately 1-50 mg/L), insulin (approximately 1-100 mg/L), sodium selenite (approximately 0.000001-0.0001 mg/L), and albumin (e.g., I) (approximately 5000-50,000 mg/L) are also present.

In some embodiments, the non-microelement components of the culture medium are determined to be present at the concentration ranges listed in the heading "Concentration Ranges in 1X Culture Medium" of Table 4. In other embodiments, the non-microelement components of the culture medium are determined to be present at the final concentrations listed in the heading "Preferred Embodiments of 1X Culture Medium" of Table 4. In other embodiments, the culture medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-microelement components of the types and concentrations listed in the heading "Preferred Embodiments of Supplements" of Table 4.

In some embodiments, the molar osmolality of the culture medium is determined to be between about 260 and 350 mOsmol. In some embodiments, the molar osmolality is determined to be between about 280 and 310 mOsmol. In some embodiments, the culture medium is determined to be supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The culture medium is determined to be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

In some embodiments, the specific culture medium described in Smith et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038/cti.2014.31) is suitable for use in this invention. In short, RPMI or CTS ^™ OpTmizer ^™ is used as the basal cell culture medium supplemented with 0, 2%, 5%, or 10% CTS ^™ immune cell serum substitutes.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, a rapid second amplification (including an amplification termed REP) is performed, and it further includes a step of selecting TILs with superior tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor reactivity.

Optionally, cell viability can be determined using standard assays known in the art after a rapid second amplification (including amplification known as REP amplification). For example, a trypan blue exclusion assay can be performed on the main TIL sample, which selectively labels dead cells and allows for viability assessment. In some embodiments, the viability of the TIL sample can be calculated and determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in a second amplification exhibit increased T cell reservoir diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, T-cell receptor (TCR) α expression is increased. In some embodiments, T-cell receptor (TCR) β expression is increased. In some embodiments, TCRab (i.e., TCRα/β) expression is increased.

In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 7.5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid secondary expansion medium (e.g., sometimes referred to as CM2 or secondary cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/culture flask of OKT-3, and 5 × ^10⁸ antigen-presenting feeder cells (APCs) as discussed in more detail below.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve vertical scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring the TILs from the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing rapid second amplification by culturing TILs in a first mini-culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the first mini-culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the TILs transferred from the first mini-culture is cultured in the second mini-culture for a period of approximately 4 to 7 days.

In some implementations, the first small-scale TIL culture is allocated to multiple TIL subpopulations of approximately 2 to 5.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 7 days; and then (b) transferring and distributing the TILs from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days.

In some implementations, the rapid second amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (a) performing rapid or second amplification by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 5 days; and then (b) transferring and distributing the TILs from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of TILs transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In some implementations, after the split of the rapid second amplification, each second container contains at least ^10⁸ TILs. In some implementations, after the split of the rapid or second amplification, each second container contains at least ^10⁸ TILs, at least ^10⁹ TILs, or at least ^10¹⁰ TILs. In one exemplary implementation, each second container contains at least ^10¹⁰ TILs.

In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2 to 5. In some implementations, the first small-scale TIL culture is allocated to multiple subpopulations of approximately 2, 3, 4, or 5.

In some embodiments, after rapid second amplification, multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid or second amplification, one or more TIL subpopulations are combined to produce a therapeutically effective amount of TIL. In some embodiments, after rapid amplification, each TIL subpopulation contains a therapeutically effective amount of TIL.

In some implementations, the rapid second amplification is performed over a period of approximately 3 to 7 days before being split into multiple steps. In some implementations, the splitting of the rapid second amplification occurs approximately day 3, day 4, day 5, day 6, or day 7 after the start of the rapid or second amplification.

In some implementations, the split of the rapid second amplification occurs approximately on days 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary implementation, the split of the rapid or second amplification occurs approximately on day 16 after the start of the first amplification.

In some implementations, rapid second amplification is performed for a period of approximately 7 to 11 days after splitting. In some implementations, rapid second amplification is performed for a period of approximately 5, 6, 7, 8, 9, 10, or 11 days after splitting.

In some embodiments, the cell culture medium used for rapid second expansion before splitting contains the same components as the cell culture medium used for rapid second expansion after splitting. In some embodiments, the cell culture medium used for rapid second expansion before splitting contains different components than the cell culture medium used for rapid second expansion after splitting.

In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by supplementing the cell culture medium from the first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is prepared by replacing the cell culture medium from the first expansion with fresh cell culture medium containing IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid secondary expansion after splitting is prepared by replacing the cell culture medium used for rapid secondary expansion before splitting with fresh culture medium containing IL-2 and OKT-3.

1. Feeder cells and antigen-presenting cells

In some embodiments, the rapid second amplification procedure described herein (e.g., including amplifications such as those described in step D of Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and those amplifications referred to as REP) requires an excess of feeder cells during REP TIL amplification and/or during rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment, and the REP procedure described in the embodiments provides an exemplary scheme for evaluating the non-replicative capability of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), then PBMCs are considered non-replicating and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In other embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is approximately 1:10, approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:125, approximately 1:150, approximately 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400, or approximately 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 100 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of approximately 7.5 × ^10⁸ feeder cells to approximately 50 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, the second expansion procedure described herein requires approximately 7.5 × ^10⁸ feeder cells to approximately 25 × ^10⁶ TILs. In other embodiments, rapid second expansion requires twice the number of feeder cells of rapid second expansion. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 5 × ^10⁸ feeder cells. In other embodiments, when initiating the first expansion as described herein requires approximately 2.5 × ^10⁸ feeder cells, the rapid second expansion requires approximately 7.5 × ^10⁸ feeder cells. In other embodiments, the rapid second expansion requires twice (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X the number of feeder cells required to initiate the first expansion.

In some embodiments, the rapid second amplification procedure described herein requires an excess of feeder cells during the rapid second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as the Ficoll-Paque gradient separation method. In some embodiments, artificial antigen-presenting cells (aAPCs) are used instead of PBMCs. In some embodiments, PBMCs are added to the rapid second amplification at twice the concentration of PBMCs added to initiate the first amplification.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the accompanying drawings and embodiments.

In some implementations, artificial antigen-presenting cells are used in place of PBMCs or in combination with PBMCs during rapid second expansion.

2. Cytokines and other additives

The rapid second amplification method described herein typically uses a culture medium containing high doses of cytokines (particularly IL-2), as is known in the art.

Alternatively, it is possible to rapidly expand TILs using combinations of cytokines, as described in U.S. Patent Application Publication No. US2017/0107490 A1, which discloses the use of combinations of two or more IL-2, IL-15, and IL-21, the contents of which are incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter having specific uses in many embodiments. The use of combinations of cytokines is particularly beneficial for generating lymphocytes, and especially T cells as described therein.

In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding OKT-3 antibody or moromuzumab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Furthermore, additives, such as peroxisome proliferator-activated receptor γ coactivator I-α agonists, including proliferator-activated receptor (PPAR)-γ agonists, such as thiazolidinedione compounds, may be used in the culture medium during step D (particularly from, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

Step E: Collect TIL

Cells can be collected after the rapid second amplification step. In some embodiments, TILs are collected after one, two, three, four or more amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL is collected after the two amplification steps (one initiating a first amplification and one rapid second amplification) provided, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

TILs can be collected in any suitable and aseptic manner, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in conjunction with the process of this invention. In some embodiments, automated systems are used to collect TILs.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. The method of the present invention can employ any cell-based collector. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (e.g., a rotating membrane or rotary filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, the rapid second amplification (e.g., step D according to Figure 8 (particularly, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

In some embodiments, step E according to FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P) is performed according to the process described herein. In some embodiments, the closed system is introduced via a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described herein is employed.

In some embodiments, TILs are collected according to the methods described herein. In some embodiments, TILs between days 14 and 16 are collected using the methods described herein. In some embodiments, TILs are collected on day 14 using the methods described herein. In some embodiments, TILs are collected on day 15 using the methods described herein. In some embodiments, TILs are collected on day 16 using the methods described herein.

Step F: Final formulation and transfer to infusion container

After steps A through E, provided in an exemplary order as shown in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and as outlined above and herein, are completed, the cells are transferred to a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In some embodiments, the TIL amplified using the methods of this disclosure is administered to the patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. The TIL amplified as disclosed herein can be administered via any suitable route known in the art. In some embodiments, the TIL is administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

VII. Other Gen 2, Gen 3 and other TIL manufacturing process implementation plans

A. PBMC feeder cell ratio

In some embodiments, the culture medium used for the amplification methods described herein (see, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) includes an anti-CD3 antibody, such as OKT-3. The combination of anti-CD3 antibody and IL-2 induces T cell activation and cell division in TIL populations. This effect is seen in full-length antibodies as well as Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

In some implementations, the number of PBMC feeder cell layers is calculated as follows:

AT cell volume (diameter 10 μm): V = (4/3) ^πr³ = 523.6 ^μm³

B. The volume of G-REX-100(M) with a height of 40 μm (4 cells): V = (4/3) ^πr³ = 4 × ^{10¹² μm³}

C. Number of cells required to fill column B: 4 × ^10¹² μm³ ^{/ 523.6 μm³ =} 7.6 × ^{10⁸ μm³} × 0.64 = 4.86 × ^10⁸

D. The optimal number of cells to activate in 4D space: 4.86 × 10⁸ ^{/ 24} = 20.25 ^{× 10⁶}

E. Extrapolation to feeder cell and TIL numbers for G-REX-500: TIL: 100 × ^10⁶ and feeder cells: 2.5 × ^10⁹

In this calculation, the approximate number of mononuclear cells required to provide TIL activation using icosahedral geometry in a cylinder with a base of 100 ^cm² is used. The experimental result for T cell threshold activation derived from the calculation is approximately 5 × ^10⁸ , which is closely correlated with NCI experimental data, as described in Jin et al., J. Immunother. 2012, 35, 283-292. In (C), the multiplier (0.64) is the random filling density of the equivalent spheres, calculated by Jaeger and Nagel, Science, 1992, 255, 1523-3. In (D), the divisor 24 is the number of equivalent spheres or “Newton number” in 4-dimensional space that can contact similar objects, as described in Musin, Russ. Math. Surv., 2003, 58, 794-795.

In some embodiments, the number of exogenously supplied antigen-presenting feeder cells during the initiation of the first expansion is approximately half the number of exogenously supplied antigen-presenting feeder cells during the rapid second expansion. In some embodiments, the method includes initiating the first expansion in a cell culture medium containing approximately 50% fewer antigen-presenting cells compared to the cell culture medium used in the rapid second expansion.

In other embodiments, the number of exogenously supplied antigen-presenting feeder cells (APCs) during the rapid second amplification is greater than the number of exogenously supplied APCs during the initiation of the first amplification.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs during the rapid second amplification period to the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, or 2.5:1. 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs during the initiation of the first amplification period is exactly or approximately ¹ ×10⁸, 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, ^1.5 × ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8×10⁸, 1.9× ^10⁸ , 2 ^{×10⁸ ,} 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3×10⁸, 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^{10⁸ ,} 3.1× ^10⁸ , or 3.2×10⁸ ^. The number of exogenously supplied APCs during the rapid second amplification period is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × 10⁸, 3.8 × ^10⁸ , 3.9 × ^10⁸ , ^{4 × 10⁸ ,} 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^10⁸ , 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , ^4.9 × ^10⁸ , 5 × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^10⁸ , 5.3 × ^10⁸ , 5.4 ^{× 10⁸ .} 5.5× ^10⁸ , 5.6× ^10⁸ , 5.7×10⁸, 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3×10⁸, 6.4× ^10⁸ , ^6.5 × ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , ^7.2 × ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , 7.8× ^10⁸ , 7.9× ^10⁸ 8× ^10⁸ , 8.1× ^10⁸ , 8.2×10⁸, 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8×10⁸, 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , ^9.7 × ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or 1×10⁹ APCs .}

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ^1.5 × 10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is selected from the range of exactly or about ² × 10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is exactly or approximately 2.5 × ^10⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification is exactly or approximately 5 × ^10⁸ APCs.

In some embodiments, the number of APCs (including, for example, PBMCs) added on day 0 of the first expansion is approximately half the number of PBMCs added on day 7 of the first expansion (e.g., day 7 of the method). In some embodiments, the method includes adding antigen-presenting cells to a first TIL population on day 0 of the first expansion and adding antigen-presenting cells to a second TIL population on day 7, wherein the number of antigen-presenting cells added on day 0 is approximately 50% of the number of antigen-presenting cells added on day 7 of the first expansion (e.g., day 7 of the method).

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is greater than the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification.

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density selected from just or about 2 × ^10⁶ APCs/ ^cm² to just or about 3 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous supply of APCs at the start of the first amplification is seeded in the culture flask at a density of just or about 2 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} Inoculate culture flasks with APCs/cm² at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4×10⁶ ^, or 4.5× ^{10⁶ .}

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from just or about 4.0 × ^10⁶ APCs/ ^cm² to just or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenously supplied APCs for rapid second amplification are seeded in culture flasks at a density selected from the range of exactly or approximately 4.0 × ^10⁶ APCs/ ^cm² .

In other implementations, the exogenously supplied APCs in the rapid second amplification are at exactly or approximately 2.5 × ^10⁶ APCs/ ^cm² , 2.6 × 10⁶ APCs/ ^cm² , 2.7 × ^10⁶ APCs/ ^cm² , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , 3.2 × ^10⁶ , 3.3 × ^10⁶ , 3.4 × ^10⁶ , 3.5 × ^10⁶ , 3.6 × ^10⁶ , 3.7 × ^10⁶ , 3.8 × ^10⁶ , 3.9 × ^10⁶ , 4 × ^10⁶ , 4.1 × ^10⁶ , 4.2 × ^10⁶ , 4.3 × ^10⁶ , 4.4 × ^10⁶ , or 4.5 × ^{10⁶ .} 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8×10⁶, 4.9× ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4×10⁶, 5.5× ^10⁶ , ^{5.6×10⁶ ,} 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , 6× ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , ^6.3 × ^10⁶ , 6.4×10⁶, 6.5× ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , ^6.8 × ^10⁶ , 6.9× ^10⁶ , 7× ^10⁶ Inoculate the culture flasks at densities of 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4× ^10⁶ , or 7.5× ^10⁶ APCs/ ^cm² .

In other implementations, the exogenous supply of APC at the start of the first amplification is exactly or approximately 1.0 × ^10⁶ , 1.1 × ^10⁶ , 1.2 × ^10⁶ , 1.3 × ^10⁶ , 1.4 × ^10⁶ , 1.5 × ^10⁶ , 1.6 × ^10⁶ , 1.7 × ^10⁶ , 1.8 × ^10⁶ , 1.9 × 10⁶, 2 × ^10⁶ , 2.1 × ^10⁶ , 2.2 × ^10⁶ , 2.3 × ^10⁶ , 2.4 × ^10⁶ , 2.5 × ^10⁶ , 2.6 × ^10⁶ , 2.7 × ^10⁶ , 2.8 × ^10⁶ , 2.9 × ^10⁶ , 3 × ^10⁶ , 3.1 × ^10⁶ , or 3.2 × ^{10⁶ .} The APCs were seeded in culture flasks at densities of 3.3× ^10⁶ , 3.4× ^10⁶ , 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4× ^10⁶ , 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , or 4.5× ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for rapid second amplification were at exactly or approximately 2.5× ^10⁶ APCs/ ^cm² , 2.6× ^10⁶ APCs/ ^cm² , 2.7× ^10⁶ APCs/ ^cm² , 2.8× ^10⁶ , 2.9× ^10⁶ , 3× ^10⁶ , and 3.1×10⁶ ^APCs/cm². 3.2× ^10⁶ , 3.3× ^10⁶ , 3.4×10⁶, 3.5× ^10⁶ , 3.6× ^10⁶ , 3.7× ^10⁶ , 3.8× ^10⁶ , 3.9× ^10⁶ , 4×10⁶, 4.1× ^10⁶ , 4.2× ^10⁶ , 4.3× ^10⁶ , 4.4× ^10⁶ , 4.5× ^10⁶ , 4.6× ^10⁶ , 4.7× ^10⁶ , 4.8× ^10⁶ , ^4.9 × ^10⁶ , 5× ^10⁶ , 5.1× ^10⁶ , 5.2× ^10⁶ , 5.3× ^10⁶ , 5.4× ^10⁶ , 5.5× ^10⁶ , ^5.6 × ^10⁶ APCs/cm² were inoculated into culture flasks at densities of 5.7× ^10⁶ , 5.8× ^10⁶ , 5.9× ^10⁶ , ⁶ × ^10⁶ , 6.1× ^10⁶ , 6.2× ^10⁶ , 6.3×10⁶, 6.4× ^10⁶ , ^6.5 × ^10⁶ , 6.6× ^10⁶ , 6.7× ^10⁶ , 6.8× ^10⁶ , 6.9×10⁶, ⁷ × ^10⁶ , 7.1× ^10⁶ , 7.2× ^10⁶ , 7.3× ^10⁶ , 7.4×10⁶, or 7.5× ^{10⁶ .}

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density selected ^from exactly or about 2.5 × ^10⁶ APCs/cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in culture flasks at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in culture flasks at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied at the start of the first amplification are seeded in the culture flask at a density selected from exactly or about 2 × ^10⁶ APCs/ ^cm² to exactly or about 3 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied at the rapid second amplification are seeded in the culture flask at a density selected from exactly or about 4 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the exogenous APCs supplied for the initial amplification are seeded in the culture flask at a density of exactly or approximately 2 × ^10⁶ APCs/ ^cm² , and the exogenous APCs supplied for the rapid second amplification are seeded in the culture flask at a density of exactly or approximately 4 × ^10⁶ APCs/ ^cm² .

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied PBMCs on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other implementations, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 2:1.

In other implementations, the ratio of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, etc. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other implementations, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first amplification is exactly or approximately 1× ^10⁸ , 1.1× ^10⁸ , 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4× ^10⁸ , 1.5× ^10⁸ , 1.6× ^10⁸ , 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9× ^10⁸ , 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , or 3.1×10⁸ ^. The exogenous supply of APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or approximately 3.5 × ^10⁸ , 3.6 × ^10⁸ , 3.7 × ^10⁸ , 3.8 × ^10⁸ , 3.9 × ^10⁸ , 4 × ^10⁸ , 4.1 × ^10⁸ , 4.2 × ^10⁸ , 4.3 × ^10⁸ , 4.4 × ^10⁸ , 4.5 × ^{10⁸ ,} 4.6 × ^10⁸ , 4.7 × ^10⁸ , 4.8 × ^10⁸ , 4.9 × ^10⁸ , ⁵ × ^10⁸ , 5.1 × ^10⁸ , 5.2 × ^{10⁸ . 8} , 5.3× ^10⁸ , 5.4× ^10⁸ , 5.5×10⁸, 5.6× ^10⁸ , 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2× ^10⁸ , 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , 6.6× ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , 7.1× ^10⁸ , 7.2× ^10⁸ , 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^{10⁸ 8} , 7.8× ^10⁸ , 7.9×10⁸, ⁸ ×10⁸, 8.1× ^10⁸ , 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7× ^10⁸ , 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , 9.6× ^10⁸ , 9.7× ^10⁸ , ^9.8 × ^10⁸ , 9.9× ^10⁸ , or 1× ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 1 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 3.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 1 × ^10⁹ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 7.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is selected from exactly or about 2 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is selected from exactly or about 4.5 × ^10⁸ APCs (including, for example, PBMCs) to exactly or about 5.5 × ^10⁸ APCs (including, for example, PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the first amplification is exactly or about 2.5 × ^10⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second amplification is exactly or about 5 × ^10⁸ APCs (including, for example, PBMCs).

In some embodiments, the number of APC (including, for example, PBMC) layers added on day 0 of the first amplification is approximately half the number of APC (including, for example, PBMC) layers added on day 7 of the rapid second amplification. In some embodiments, the method includes adding antigen-presenting cell layers to a first TIL population on day 0 of the first amplification and adding antigen-presenting cell layers to a second TIL population on day 7, wherein the number of antigen-presenting cell layers added on day 0 is approximately 50% of the number of antigen-presenting cell layers added on day 7.

In other implementations, the number of exogenously supplied APC (including, for example, PBMC) layers on day 7 of the rapid second amplification is greater than the number of exogenously supplied APC (including, for example, PBMC) layers on day 0 of the initiation of the first amplification.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about three cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1.5 to just or about 2.5 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about two cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers, and day 7 of the rapid second amplification occurs with an average thickness of just or approximately 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3. It occurs in the presence of layered APCs (including, for example, PBMCs) of 8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1 cell layer to just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 to just or about 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 1, 2, or 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9, or 10 cell layers.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from the range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is exactly or approximately 1:2.

In other embodiments, day 0 of the first amplification occurs in the presence of a layered APC (including, for example, PBMC) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMC), and day 7 of the rapid second amplification occurs in the presence of a layered APC (including, for example, PBMC) having a second average thickness equal to the number of layers of the second APC (including, for example, PBMC), wherein the ratio of the number of layers of the first APC (including, for example, PBMC) to the number of layers of the second APC (including, for example, PBMC) is selected from... Exactly or approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1 :4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.0 × ^10⁶ APCs/ ^cm² to about 4.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 2.5 × ^10⁶ APCs/ ^cm² to about 7.5 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 1.5 × ^10⁶ APCs/ ^cm² to about 3.5 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 3.5 × ^10⁶ APCs/ ^cm² to about 6.0 × ^10⁶ APCs/ ^cm² .

In some implementations, the number of APCs in the initial amplification is selected from the range of about 2.0 × ^10⁶ APCs/ ^cm² to about 3.0 × ^10⁶ APCs/ ^cm² , and the number of APCs in the rapid second amplification is selected from the range of about 4.0 × ^10⁶ APCs/ ^cm² to about 5.5 × ^10⁶ APCs/ ^cm² .

A. Optional cell culture medium components

1. Anti-CD3 antibody

In some embodiments, the culture medium used for the amplification methods described herein (including an amplification method known as REP, see, for example, Figures 1 and 8 (especially, for example, Figure 8B)) includes an anti-CD3 antibody. The combination of the anti-CD3 antibody and IL-2 induces T cell activation and cell division in a TIL population. This effect can be seen with both full-length antibodies and Fab and F(ab')2 fragments, the former generally preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is hereby incorporated in its entirety by reference.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California). See Table 1.

As those skilled in the art will appreciate, suitable anti-human CD3 antibodies can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from a variety of mammals, including (but not limited to) rodent, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody morocumab is used (available from Ortho-McNeil, Laritan, NJ, or Miltenyi Biotech, Auburn, California).

2,4-1BB (CD137) agonist

In some embodiments, the cell culture medium initiating the first amplification and/or rapid second amplification contains a TNFRSF agonist. In some embodiments, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule may contain any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The 4-1BB agonist or 4-1BB binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies); monoclonal antibodies (including full-length monoclonal antibodies); polyclonal antibodies; multispecific antibodies (e.g., bispecific antibodies); human, humanized, or chimeric antibodies; and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to 4-1BB, such as scFv molecules. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the 4-1BB agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the 4-1BB agonist used in the methods and compositions disclosed in this invention includes anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectin, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. Agonistant anti-4-1BB antibodies are known to induce strong immune responses. (Lee et al., PLOSOne 2013, 8, e69677). In some embodiments, the 4-1BB agonist is an agonistant anti-4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In some embodiments, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utumumab, or urilumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

In some embodiments, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In some embodiments, polymeric 4-1BB agonists, such as trimeric or hexameric 4-1BB agonists (having three or six ligand-binding domains), can induce the formation of desirable receptor (4-1BBL) clusters and internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins containing three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic 4-1BB antibodies and fusion proteins induce strong immune responses. In some embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO:40) with high affinity and agonistic activity. In some embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO:40). In some embodiments, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO:41). The amino acid sequences of the 4-1BB antigens bound by the 4-1BB agonist or binding molecule are summarized in Table 5.

Table 5.4-1 shows the amino acid sequence of the BB antigen.

In some embodiments, the described compositions, processes, and methods include a 4-1BB agonist that binds to human or rodent 4-1BB at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at _{a KD} of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include 4-1BB agonists that _bind to human or rodent 4-1BB at a k-association rate of about 7.5 × ^{10⁵ 1} /M·s or faster, at a k _-association rate of about 7.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ 1/M·s or faster, at a k- _association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include binding to human or rodent 4-1BB at a k _{-dissociation} rate of about 2 × ^10⁻⁵ 1/s or _slower , at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about _2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 × ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/s or _slower , at about 2.6 × ^10⁻⁵ 1/s or _slower , or at about 2.7 × ^10⁻⁵ 1/s or slower. 4-1BB agonists that bind to human or rodent 4-1BB at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include a _4-1BB agonist that binds to human or rodent 4-1BB at an _IC50 of about ₁₀ nM or less, at about 9 nM or less, at about ₈ nM or less, at about 7 nM or less, at about ₆ nM or less, at about ₅ nM or less, at about ₄ nM or less, at about 3 nM or less, at about ₂ nM or less, or at about 1 nM or less _{. 50} combines with 4-1BB in humans or rodents.

In some embodiments, the 4-1BB agonist is utumumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant, or biosimilar thereof. Utumumab is available from Pfizer. Utumumab is an immunoglobulin G2-λ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of utumumab is illustrated in Table 6. Utumumab contains glycosylation sites at Asn59 and Asn292; intrachain disulfide bridges in the heavy chain at positions 22-96 ( _VH - _VL ), 143-199 ( _CH1 - _CL ), 256-316 ( _CH2 ), and 362-420 ( _CH3 ); and intrachain disulfide bridges at positions 22'-87' ( _VH - _VL ) and 136'-195' ( _CH1 -CL _). Intrachain disulfide bridges of the light chain; interchain heavy chain-heavy chain disulfide bridges located at IgG2A isoform positions 218-218, 219-219, 222-222 and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222 and 225-225 and at IgG2B isoform positions 219-130(2), 222-222 and 225-225; and interchain heavy chain-light chain disulfide bridges located at IgG2A isoform positions 130-213'(2), IgG2A/B isoform positions 218-213' and 130-213' and at IgG2B isoform positions 218-213'(2). The preparation and characterization of utumumab, as well as its variants and fragments, are described in U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678 and International Patent Application Publication No. WO 2012/032433 A1, the disclosures of which are incorporated herein by reference. The preclinical characteristics of utumumab are described in Fisher et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utumumab in various hematologic and solid tumor indications include US National Institutes of Health (NIH) clinicaltrials.gov identification numbers NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:42 and the light chain provided in SEQ ID NO:43. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utumumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:44, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:45, along with its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of utumumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity, such as 97%, 98%, 99%, or 100%, with an amino acid sequence that is 97%, 98%, 99%, or 100% sequence identity with a reference drug or reference biological product, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, which is utumumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, which is utumumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab.

Table 6. Amino acid sequences of 4-1BB agonist antibodies associated with utumumab.

In some embodiments, the 4-1BB agonist is the monoclonal antibody urogenumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biosimilar thereof. Ureogenumab is available from Bristol-Myers Squibb and Creative Biolabs. Ureogenumab is an immunoglobulin G4-κ anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)] Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of urogenumab is illustrated in Table 7. Urelimab contains N-glycosylation sites at positions 298 (and 298”); intrachain disulfide bridges in the heavy chain at positions 22–95 ( _VH – _VL ), 148–204 ( _CH1 – _CL ), 262–322 ( _CH2 ), and 368–426 ( _CH3 ) (and at positions 22”–95”, 148”–204”, 262”–322”, and 368”–426”); and intrachain disulfide bridges in the heavy chain at positions 23”–88” ( _VH – _VL ) and 136”–196” ( _CH1 –CL _). (and intra-chain disulfide bridges at positions 23”'-88”' and 136”'-196”'); inter-chain heavy chain-heavy chain disulfide bridges at positions 227-227” and 230-230”; and inter-chain heavy chain-light chain disulfide bridges at positions 135-216”' and 135”-216”'. The preparation and characterization of urogenumab, as well as its variants and fragments, are described in U.S. Patent Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical characteristics of urogenumab are described in Segal et al., Clin. Cancer. Res. 2016, available at http://dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical trials of ureglucumab in various hematologic and solid tumor indications include NCT01775631, NCT02110082, NCT02253992, and NCT01471210 from the National Institutes of Health (NIH) clinicaltrials.gov.

In some embodiments, the 4-1BB agonist comprises the heavy chain provided in SEQ ID NO:52 and the light chain provided in SEQ ID NO:53. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urinumab. In some embodiments, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:54, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:55, and its conserved amino acid substitutions. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising _VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively.

In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of urogenumab approved by a regulatory agency. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is urogenumab. The 4-1BB agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is uroreglumab.

Table 7. Amino acid sequences of 4-1BB agonist antibodies associated with urilumab.

In some embodiments, the 4-1BB agonist is selected from the group consisting of: 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), produced by a cell line registered as ATCC No. HB-11248 and disclosed in U.S. Patent No. 6,974,863. Antibodies disclosed in the following countries include: 5F4 (BioLegend 31 1503), C65-485 (BDPharmingen 559446), antibodies disclosed in US Patent Application Publication No. 2005/0095244, antibodies disclosed in US Patent No. 7,288,638 (e.g., 20H4.9-IgG1 (BMS-663031)), antibodies disclosed in US Patent No. 6,887,673 (e.g., 4E9 or BMS-554271), and antibodies disclosed in US Patent No. 7,214,493. Antibodies disclosed in U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, U.S. Patent No. 6,905,685 (e.g., 4E9 or BMS-554271), U.S. Patent No. 6,362,325 (e.g., 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), U.S. Patent No. 6,974,863 (e.g., 53A2), and U.S. Patent No. 6,210,669 (e.g., 5E9 or 5E2). The antibodies described in U.S. Patent No. 5,928,893 (1D8, 3B8, or 3E1), U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, and International Patent Application Publications Nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, as well as fragments, derivatives, conjugates, variants, or bioanalytes thereof, wherein the disclosure of each of the foregoing patents or patent applications is incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein described in the following: International Patent Application Publications No. WO 2008/025516 A1, WO 2009/007120A1, WO 2010/003766 A1, WO 2010/010051 A1 and WO 2010/078966 A1; U.S. Patent Application Publication No. US 201 The disclosures of U.S. Patent Nos. 1/0027218A1, 2015/0126709 A1, 2011/0111494A1, 2015/0110734 A1, and 2015/0126710 A1; and U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, are incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic fusion protein as depicted in structure IA (C-terminal Fc antibody fragment fusion protein) or structure IB (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof (see Figure 18). In structures IA and IB, the cylinder refers to an individual polypeptide-binding domain. Structures IA and IB contain three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL (4-1BB ligand, CD137 ligand (CD137L), or tumor necrosis factor superfamily member 9 (TNFSF9)) or antibodies binding to 4-1BB. These TNFRSF binding domains fold to form a trivalent protein, which is then linked to a second trivalent protein via an IgG1-Fc (including _CH3 and _CH2 domains). The IgG1-Fc then serves to link the two trivalent proteins together via disulfide bonds (elongated ellipses), thereby stabilizing the structure and providing an agonist capable of linking the intracellular signaling domains of six receptors to signaling proteins to form a signaling complex. A TNFRSF binding domain, represented as a cylinder, may be an scFv domain containing, for example, _VH and _VL chains linked by a linker, which may contain hydrophilic residues and provide flexibility with Gly and Ser sequences, and solubility with Glu and Lys. Any scFv domain design can be used, such as those described in: de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad et al., Clin. & Dev. Immunol. 2012, 980250; Monnier et al., Antibodies, 2013, 2, 193-208; or other references incorporated herein by reference. The structure of this form of fusion protein is described in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of structures I-A provided in Figure 18 are shown in Table 8. The Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO: 62), a complete hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). Preferred adapters for linking C-terminal Fc antibodies are selected from the embodiments provided in SEQ ID NO: 63 to SEQ ID NO: 72, including adapters suitable for fusing other polypeptides.

Table 8. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with C-terminal Fc-antibody fragment fusion protein design (structure I-A).

The amino acid sequences of the other polypeptide domains of structures I-B provided in Figure 18 are shown in Table 9. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SED ID NO:74 to SEQ ID NO:76.

Table 9. Amino acid sequences of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) with N-terminal Fc-antibody fragment fusion protein design (structures I-B).

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B includes one or more 4-1BB binding domains selected from the group consisting of: variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains of utumumab, variable heavy and variable light chains selected from the variable heavy and variable light chains described in Table 10, any combination of the aforementioned variable heavy and variable light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:77. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a soluble 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO:78.

In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:43 and SEQ ID NO:44, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively, wherein the _VH and _VL domains are connected by linkers. In some embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, said one or more binding domains being scFv domains comprising _VH and _VL regions, each at least 95% identical to the _VH and _VL sequences provided in Table 10, wherein the _VH and _VL domains are connected by linkers.

Table 10. Applicable to the 4-1BB binding domain in fusion proteins or other polypeptide domains applicable to scFv 4-1BB agonist antibodies.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stem region (which promotes trimerization and provides a distance from the cell membrane, but is not part of the 4-1BB binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some embodiments, the 4-1BB agonist is a BPS Bioscience 4-1BB agonist antibody, catalog number 79097-2, available from BPS Bioscience in San Diego, California, USA. In other embodiments, the 4-1BB agonist is a Creative Biolabs 4-1BB agonist antibody, catalog number MOM-18179, available from Creative Biolabs in Shirley, NY, USA.

3. OX40 (CD134) agonist

In some embodiments, the TNFRSF agonist is an OX40 (CD134) agonist. The OX40 agonist can be any OX40-binding molecule known in the art. The OX40-binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX40. The OX40 agonist or OX40-binding molecule may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The OX40 agonist or OX40-binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized or chimeric antibodies, and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above substances, and engineered forms of antibodies binding to OX40, such as scFv molecules. In some embodiments, the OX40 agonist is an antigen-binding protein of a fully human antibody. In some embodiments, the OX40 agonist is an antigen-binding protein of a humanized antibody. In some embodiments, the OX40 agonist used in the methods and compositions of this disclosure includes anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 adenettin, anti-OX40 domain antibodies, single-chain anti-OX40 fragments, heavy-chain anti-OX40 fragments, light-chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. In some embodiments, the OX40 agonist is an agonistic anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

In some embodiments, the OX40 agonist or OX40 binding molecule may also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun et al., J. Immunother. 2009, 182, 1481-89. In some embodiments, poly-OX40 agonists, such as trimeric or hexameric OX40 agonists (having three or six ligand-binding domains), can induce clustering of the preferred receptor (OX40L) and the formation of internal cell signaling complexes, compared to agonistic monoclonal antibodies that typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic OX40 antibodies and fusion proteins can induce strong immune responses. (Curti et al., Cancer Res. 2013, 73, 7189-98). In some embodiments, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficiently reducing toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC), such as NK cell toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the OX40 agonist is characterized by binding to human OX40 (SEQ ID NO: 85) with high affinity and agonistic activity. In some embodiments, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 85). In some embodiments, the OX40 agonist is a binding molecule that binds to mouse OX40 (SEQ ID NO: 86). Table 11 summarizes the amino acid sequences of the OX40 antigen bound to the OX40 agonist or binding molecule.

Table 11. Amino acid sequence of OX40 antigen.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind human or rodent OX40 at a _KD of about 100 pM or less, at a _KD of about 90 pM or less, at a _KD of about 80 pM or less, at a _KD of about 70 pM or less, at a _KD of about 60 pM or less, at a _KD of about 50 pM or less, at a _KD of about 40 pM or less, or at a _KD of about 30 pM or less.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that _bind to human or rodent OX40 at a k-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 7.5 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8 × ^10⁵ 1/M·s or faster, at a k _-association rate of about 8.5 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9 × 10⁵ ¹ /M·s or faster, at a k _-association rate of about 9.5 × ^10⁵ 1/M·s or faster, or at a k- _association rate of about 1 × ^10⁶ 1/M·s or faster.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at a k _{-dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, at about 2.1 × ^10⁻⁵ 1/s or _slower , at about 2.2 × ^10⁻⁵ 1/s or slower, at about 2.3 × ^10⁻⁵ 1/s or slower, at about 2.4 _× ^10⁻⁵ 1/s or _slower , at about 2.5 × ^10⁻⁵ 1/ _s or slower, and at about 2.6 × ^10⁻⁵ 1/s or slower. It binds to human or rodent OX40 at a k _dissociation of 1/s or slower, at a k _dissociation of about 2.7 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.8 × ^10⁻⁵ 1/s or slower, at a k _dissociation of about 2.9 × ^10⁻⁵ 1/s or slower, or at a k _dissociation of about 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the described compositions, processes, and methods include OX40 agonists that bind to human or rodent OX40 at an _IC50 of about 10 nM or less, at an IC50 of about 9 nM or less, at an _IC50 of about 8 nM or less _, at an _IC50 of about 7 nM or less, at an IC50 of about 6 nM or less, at an _IC50 of about ₅ nM or less, at an IC50 of about 4 nM or less, at an _IC50 of about 3 nM or less, at _{an IC50} of about 2 nM or less, or at an _IC50 of about 1 nM or less.

In some implementations, the OX40 agonist is tavoricumab, also known as MEDI 0562 or MEDI-0562. Tavoricumab is available from MedImmune, a subsidiary of AstraZeneca. Tavoricumab is a humanized and chimeric monoclonal antibody against immunoglobulin G1-κ [Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)]. The amino acid sequence of tavoricumab is illustrated in Table 12. Tavocitumab contains N-glycosylation sites at positions 301 and 301", with fucosylated complex biantennary CHO-type glycans; intrachain disulfide bridges in the heavy chain at positions 22-95 ( _VH - _VL ), 148-204 ( _CH1 - _CL ), 265-325 ( _CH2 ), and 371-429 ( _CH3 ) (and at positions 22"-95", 148"-204", 265"-325" and 371"-429"); and intrachain disulfide bridges in the heavy chain at positions 23"-88" ( _VH - _VL ) and 134"-194" ( _CH1 -CL ₎ . Intra-chain disulfide bridges of the light chain at positions 23”'-88”' and 134”'-194”'; inter-chain heavy chain-heavy chain disulfide bridges at positions 230-230” and 233-233”'; and inter-chain heavy chain-light chain disulfide bridges at positions 224-214”' and 224”-214”'. Current clinical trials of tavoricumab in various solid tumor indications include NCT02318394 and NCT02705482 from the National Institutes of Health clinicaltrials.gov.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:87 and the light chain provided in SEQ ID NO:88. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of tavoricumab. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:89, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:90, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90 _, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises an scFv antibody, said scFv antibody comprising _VH and _VL regions _, each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of the OX40 agonist approved by a regulatory agency, referencing tavoricumab. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is tavoricumab.

Table 12. Amino acid sequences of OX40 agonist antibodies associated with tavoricumab.

In some embodiments, the OX40 agonist is 11D4, a fully human antibody available from Pfizer. The preparation and characterization of 11D4 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is set forth in Table 13.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:97 and the light chain provided in SEQ ID NO:98. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:99, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:100, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _{VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises VH and VL regions} , each at least 98% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100 _, respectively. In some embodiments, the OX40 agonist comprises _VH and VL _regions , each at least 97% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody approved by a regulatory agency with reference to 11D4. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug product or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 11D4.

Table 13. Amino acid sequences of OX40 agonist antibodies associated with 11D4.

In some embodiments, the OX40 agonist is 18D8, a fully human antibody available from Pfizer. The preparation and characterization of 18D8 are described in U.S. Patents 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is set forth in Table 14.

In some embodiments, the OX40 agonist comprises the heavy chain provided in SEQ ID NO:107 and the light chain provided in SEQ ID NO:108. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO:108, respectively.

In some embodiments, the OX40 agonist comprises a heavy chain and a light chain CDR or variable region (VR) of 18D8. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:109, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:110, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO: ₁₁₀ , respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to 18D8. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence having at least 97%, such as 97%, 98%, 99%, or 100%, of a reference drug product or reference biological product, and comprising one or more post-translational modifications relative to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 18D8.

Table 14. Amino acid sequences of OX40 agonist antibodies associated with 18D8.

In some embodiments, the OX40 agonist is Hu119-122, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu119-122 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is set forth in Table 15.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:117, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:118 and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 96% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 95% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu119-122. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122.

Table 15. Amino acid sequences of OX40 agonist antibodies associated with Hu119-122.

In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu106-222 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is set forth in Table 16.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222. In some embodiments, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:125, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO:126, and its conserved amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 96% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126. In some embodiments, the OX40 agonist comprises _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences and conserved amino acid substitutions described in SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, respectively.

In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu106-222. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. The OX40 agonist antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222.

Table 16. Amino acid sequences of OX40 agonist antibodies associated with Hu106-222.

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B12). MEDI6469 is a murine monoclonal antibody. (Weinberg et al., J. Immunother. 2006, 29, 575-585). In some embodiments, the OX40 agonist is an antibody produced from a 9B12 hybridoma and hosted by Biovest Corporation (Malvern, MA, USA), as described in Weinberg et al., J. Immunother. 2006, 29, 575-585, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the antibody comprises the CDR sequence of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of MEDI6469.

In some embodiments, the OX40 agonist is L106 BD (Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the CDR of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106 (BD Pharmingen, product #340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the CDR of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some implementations, the OX40 agonist is a murine monoclonal antibody anti-mCD134/mOX40 (clone OX86), available from BioXcell's InVivoMAb in West Lebanon, New Hampshire.

In some embodiments, the OX40 agonist is selected from the OX40 agonists described below: International Patent Application Publications WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191 and WO 2014/148895; European Patent Application EP 0672141; US Patent Application Publication US2010/136030, ... US Nos. 2014/377284, 2015/190506, and 2015/132288 (including clones 20E5 and 12H3); and US U.S. Patents 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the OX40 agonist is an OX40 agonistic fusion protein as depicted in structures I-A (C-terminal Fc-antibody fragment fusion protein) or I-B (N-terminal Fc-antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof. The characteristics of structures I-A and I-B have been described above and in U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequences of the polypeptide domains of structure I-A provided in Figure 18 are shown in Table 9. The Fc domain preferably comprises an intact constant domain (amino acids 17-230 of SEQ ID NO: 62), an intact hinge domain (amino acids 1-16 of SEQ ID NO: 62), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). The preferred adapter for linking the C-terminal Fc antibody can be selected from the embodiments provided in SEQ ID NO:63 to SEQ ID NO:72, including adapters suitable for fusing other peptides. Similarly, the amino acid sequences of the polypeptide domains of structures I-B provided in Figure 18 are shown in Table 10. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein in structures I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:73, and the adapter sequence is preferably selected from those embodiments set forth in SEQ ID NO:74 to SEQ ID NO:76.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40 binding domains selected from the group consisting of: the variable heavy and light chains of tavocisumab, the variable heavy and light chains of 11D4, the variable heavy and light chains of 18D8, the variable heavy and light chains of Hu119-122, the variable heavy and light chains of Hu106-222, the variable heavy and light chains selected from the variable heavy and light chains described in Table 17, any combination of the aforementioned variable heavy and light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 133. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the soluble OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 134. In some embodiments, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence according to SEQ ID NO: 135.

In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:109 and SEQ ID NO:110, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains including _VH and _VL regions, each respectively at least 95% identical to the sequences shown in SEQ ID NO:127 and SEQ ID NO:128, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and _VL regions, respectively, that are at least 95% identical to the sequences shown in SEQ ID NO: 125 and SEQ ID NO: 126, wherein the _VH and _VL regions are connected by linkers. In some embodiments, the OX40 agonist fusion protein according to structure IA or IB includes one or more OX40 binding domains, said one or more binding domains being scFv domains, said scFv domains containing _VH and VL regions, respectively, that are at least 95% identical to the _VH and _VL sequences provided in Table 17, wherein the _VH and _{VL regions} are connected by linkers.

Table 17. OX40 binding domains suitable for use as fusion proteins (e.g., structures I-A and I-B) or other polypeptide domains suitable for use as scFv OX40 agonist antibodies.

In some embodiments, the OX40 agonist is an OX40-agonistic single-chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain further comprising an additional domain at an N-terminus and/or a C-terminus, wherein said additional domain is a Fab or Fc fragment domain. In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble OX40-binding domain; (ii) a first peptide linker; (iii) a second soluble OX40-binding domain; (iv) a second peptide linker; and (v) a third soluble OX40-binding domain further comprising additional domains at the N-terminus and/or C-terminus, wherein said additional domains are Fab or Fc fragment domains, wherein each of the soluble OX40-binding domains lacks a stem region (which facilitates trimerization and provides a distance from the cell membrane, but is not part of the OX40-binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In some embodiments, the OX40 agonist is an OX40-activating single-chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain; (ii) a first peptide linker; (iii) a second soluble TNF superfamily cytokine domain; (iv) a second peptide linker; and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily cytokine domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonist fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In some embodiments, the OX40 agonist is an OX40-agonistic scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In some implementations, the OX40 agonist is Creative Biolabs' OX40 agonist monoclonal antibody MOM-18455, which is available from Creative Biolabs in Sherry, New York, USA.

In some implementations, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, which is available from BioLegend, Inc., San Diego, California, USA.

B. Optional cell viability analysis

Optionally, after initiating the first expansion (sometimes referred to as initial bulk expansion), cell viability can be determined using standard assays known in the art. Therefore, in some embodiments, the method includes performing cell viability determination after initiating the first expansion. For example, a trypan blue exclusion assay can be performed on the bulk TIL sample, which selectively labels dead cells and allows for viability assessment. Other assays used to test viability may include (but are not limited to) an alamar blue assay and an MTT assay.

1. Cell counting, viability, and flow cytometry

In some embodiments, cell count and/or viability are measured. Expression of biomarkers (e.g., but not limited to CD3, CD4, CD8, and CD56, and any other biomarkers disclosed or described herein) can be measured by flow cytometry using a FACSCanto ^™ flow cytometer (BD Biosciences), with antibodies, such as (but not limited to) those available from BD Biosciences (BD Biosciences, San Jose, CA). Cells can be manually counted using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art (including, but not limited to, trypan blue staining). Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0282694, which is incorporated herein by reference in its entirety. Cell viability can also be determined based on U.S. Patent Application Publication No. 2018/0280436 or International Patent Application Publication No. WO/2018/081473, both of which are incorporated herein by reference in their entirety for all purposes.

In some cases, the primary TIL population can be immediately cryopreserved using the methods discussed below. Alternatively, the primary TIL population can undergo re-expansion (REP) and then be cryopreserved as discussed below. Similarly, in cases where genetically modified TILs will be used in therapy, the primary or REP TIL population can be genetically modified for appropriate treatment.

2. Cell Culture

In some embodiments, the method for amplifying TILs (including those discussed above and illustrated in Figures 1 and 8, particularly those exemplified in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) may include the use of about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the culture medium is serum-free. In some embodiments, the culture medium used to initiate the first amplification is serum-free. In some embodiments, the culture medium used to initiate the second amplification is serum-free. In some embodiments, the culture medium used to initiate both the first and second amplifications (also referred to as rapid second amplification) is serum-free. In some embodiments, TIL number amplification uses no more than one type of cell culture medium. Any suitable cell culture medium can be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, CA). In this regard, the method of the present invention advantageously reduces the amount and number of culture media types required for TIL number amplification. In some embodiments, TIL number amplification may include feeding cells at a frequency not exceeding once every three or four days. Amplifying cell numbers in an aerated container simplifies the procedure required for cell number amplification by reducing the feeding frequency needed.

In some embodiments, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME)-free.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for a period of about 1 to 8 days, for example, about 7 days as the initiation of the first amplification, or about 8 days as the initiation of the first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for a period of about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for about 1 to 7 days (e.g., about 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TILs to a second ventilated container and amplifying the number of TILs in the second ventilated container for about 7 to 14 days or about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days, about 10 days, or about 11 days), the second ventilated container containing a cell culture medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container for approximately 1 to 7 days (e.g., approximately 7 days) as the initiation of a first amplification, the first ventilated container containing a cell culture medium comprising IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring the TIL to a second ventilated container and amplifying the number of TILs in the second ventilated container for approximately 7 to 11 days (e.g., approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, or approximately 11 days), the second ventilated container containing a cell culture medium comprising IL-2, 2X antigen-presenting feeder cells, and OKT-3.

In some embodiments, TILs are amplified in a breathable container. Breathable containers have been used to amplify TILs, using PBMCs, and employing methods, compositions, and apparatus known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In some embodiments, TILs are amplified in a breathable bag. In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the Xuri Cell Expansion System W25 (GE Healthcare)). In some embodiments, TILs are amplified using a cell expansion system for amplifying TILs in a breathable bag (e.g., the WAVE bioreactor system, also known as the Xuri Cell Expansion System W5 (GE Healthcare)). In some embodiments, the cell expansion system includes a breathable cell bag with a volume selected from the group consisting of: about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

In some embodiments, TILs can be expanded in G-REX culture flasks (available from Wilson Wolf Manufacturing). Such embodiments expand the cell population from approximately 5 × ^10⁵ cells/cm² to 10 × ^10⁶ to 30 × ^10⁶ cells/cm². In some embodiments, this is not performed feedering. In some embodiments, this is not performed feedering, as long as the culture medium in the G-REX culture flask is at a height of approximately 10 cm. In some embodiments, this is not performed feedering, but one or more cytokines are added. In some embodiments, the cytokines are added as a bolus, without needing to be mixed with the culture medium. Such containers, apparatuses, and methods are known in the art and have been used to amplify TILs, and include those described in the following: U.S. Patent Application Publication No. US2014/0377739A1, International Publication No. WO 2014/210036A1, U.S. Patent Application Publication No. US 2013/0115617A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Patent No. US 8,809,050 B2, International Publication No. WO 2011/072088A2, U.S. Patent Application Publication No. US2016/0208216A1, U.S. Patent Application Publication No. US2012/0244133 A1, International Publication No. WO 2012/129201A1, and U.S. Patent Application Publication No. US2013/0102075. The disclosures of U.S. Patent No. A1, U.S. Patent No. 8,956,860 B2, International Publication No. WO 2013/173835A1, and U.S. Patent Application Publication No. US2015/0175966 A1 are incorporated herein by reference. Similar processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

Optional gene knockdown or gene knockout in C.TIL

In some embodiments, the amplified TILs of the present invention are further manipulated before, during, or after the amplification step, including during a closed, aseptic manufacturing process (each as provided herein), to temporarily alter protein expression. In some embodiments, the temporarily altered protein expression is due to temporary gene editing. In some embodiments, the amplified TILs of the present invention are treated with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in TILs. In some embodiments, TFs and/or other molecules capable of temporarily altering protein expression provide altered tumor antigen expression and/or altered number of tumor antigen-specific T cells in the TIL population.

In some embodiments, the method includes gene editing of a TIL population. In some embodiments, the method includes gene editing of a first TIL population, a second TIL population, and/or a third TIL population.

In some embodiments, the invention includes gene editing via nucleotide insertion, such as via ribonucleic acid (RNA) insertion, including the insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA) into a TIL population to promote or inhibit the expression of one or more proteins, as well as a combination of promoting one group of proteins and inhibiting another group of proteins.

In some embodiments, the amplified TILs of the present invention undergo a temporary alteration of protein expression. In some embodiments, the temporary alteration of protein expression occurs in the host TIL population prior to the first amplification, including, for example, the TIL population obtained from step A as indicated in, for example, Figure 8 (particularly Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs during the first amplification, including, for example, the TIL population obtained from step B as indicated in Figure 8 (e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the temporary alteration of protein expression occurs after the first amplification, including, for example, the TIL population transitioning between the first and second amplifications (e.g., the second TIL population described herein), the TIL population obtained from, for example, step B as indicated in Figure 8, and included in step C. In some embodiments, the temporary alteration of protein expression occurs in the main TIL population prior to the second amplification, including, for example, the TIL population obtained from, for example, step C as indicated in Figure 8, and prior to its amplification in step D. In some embodiments, the temporary alteration of protein expression occurs during a second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8 (e.g., a third TIL population). In some embodiments, the temporary alteration of protein expression occurs after the second amplification, including, for example, in a TIL population amplified in step D as indicated in Figure 8.

In some embodiments, the method for temporarily altering protein expression in a TIL population includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. In some embodiments, the method for temporarily altering protein expression in a TIL population includes a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for temporarily altering protein expression in a TIL population include transfection steps using methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference.

In some embodiments, temporary alteration of protein expression induces an increase in stem memory T cells (TSCMs). TSCMs are early progenitor cells of antigen-experiencing central memory T cells. TSCMs typically exhibit the long-term survival, self-renewal, and pluripotent capabilities that define stem cells and are generally required for the production of effective TIL products. In mouse models of adoptive cell transfer, enhanced antitumor activity of TSCMs compared to other subsets of T cells has been demonstrated. In some embodiments, temporary alteration of protein expression induces a TIL population with a composition containing a high proportion of TSCMs. In some embodiments, temporary alteration of protein expression induces an increase in the percentage of TSCMs of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, a temporary alteration of protein expression causes a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCM in a TIL population. In some embodiments, a temporary alteration of protein expression causes a TIL population with at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM. In some implementations, a temporary alteration in protein expression results in a therapeutic TIL population having at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM.

In some implementations, a temporary alteration of protein expression induces antigen-experiencing T-cell rejuvenation. In some implementations, rejuvenation includes, for example, increased proliferation, increased T-cell activation, and/or increased antigen recognition.

In some implementations, temporarily altering protein expression changes the expression of a large portion of T cells to preserve the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression does not alter the tumor-derived TCR reservoir. In some implementations, temporarily altering protein expression maintains the tumor-derived TCR reservoir.

In some implementations, temporary alteration of a protein induces a change in the expression of a specific gene. In some implementations, the temporary alteration of protein expression targets genes including (but not limited to) the following: PD-1 (also known as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, chimeric co-stimulatory receptor (CCR), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, and CC. R5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some implementations, protein expression targeting genes selected from the following groups is temporarily altered: PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL. 2(MCP-1), CCL3(MIP-1α), CCL4(MIP1-β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high migration group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR), and/or cAMP protein kinase A (PKA). In some embodiments, protein expression is temporarily altered to target PD-1. In some embodiments, protein expression is temporarily altered to target TGFBR2. In some embodiments, protein expression is temporarily altered to target CCR4/5. In some embodiments, protein expression is temporarily altered to target CBL-B. In some embodiments, protein expression is temporarily altered to target CISH. In some embodiments, protein expression is temporarily altered to target CCR (chimeric co-stimulatory receptor). In some embodiments, protein expression is temporarily altered to target IL-2. In some embodiments, protein expression is temporarily altered to target IL-12. In some embodiments, protein expression targeting IL-15 is temporarily altered. In some embodiments, protein expression targeting IL-21 is temporarily altered. In some embodiments, protein expression targeting NOTCH 1/2 ICD is temporarily altered. In some embodiments, protein expression targeting TIM3 is temporarily altered. In some embodiments, protein expression targeting LAG3 is temporarily altered. In some embodiments, protein expression targeting TIGIT is temporarily altered. In some embodiments, protein expression targeting TGFβ is temporarily altered. In some embodiments, protein expression targeting CCR1 is temporarily altered. In some embodiments, protein expression targeting CCR2 is temporarily altered. In some embodiments, protein expression targeting CCR4 is temporarily altered. In some embodiments, protein expression targeting CCR5 is temporarily altered. In some embodiments, protein expression targeting CXCR1 is temporarily altered. In some embodiments, protein expression targeting CXCR2 is temporarily altered. In some embodiments, protein expression targeting CSCR3 is temporarily altered. In some embodiments, protein expression targeting CCL2 (MCP-1) is temporarily altered. In some embodiments, protein expression targeting CCL3 (MIP-1α) is temporarily altered. In some embodiments, protein expression targeting CCL4 (MIP1-β) is temporarily altered. In some embodiments, protein expression targeting CCL5 (RANTES) is temporarily altered. In some embodiments, protein expression targeting CXCL1 is temporarily altered. In some embodiments, protein expression targeting CXCL8 is temporarily altered. In some embodiments, protein expression targeting CCL22 is temporarily altered. In some embodiments, protein expression targeting CCL17 is temporarily altered. In some embodiments, protein expression targeting VHL is temporarily altered. In some embodiments, protein expression targeting CD44 is temporarily altered. In some embodiments, protein expression targeting PIK3CD is temporarily altered. In some embodiments, protein expression targeting SOCS1 is temporarily altered. In some embodiments, protein expression targeting thymocyte selection-associated high migration group (HMG) box (TOX) is temporarily altered. In some embodiments, protein expression targeting ankyrin repeat domain 11 (ANKRD11) is temporarily altered. In some embodiments, protein expression targeting BCL6 co-inhibitor (BCOR) is temporarily altered. In some embodiments, protein expression targeting cAMP protein kinase A (PKA) is temporarily altered.

In some embodiments, a temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors. In some embodiments, the chemokine receptors overexpressed due to temporary protein expression include receptors with ligands, said ligands including (but not limited to) CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.

In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and/or TGFβ (including resulting in, for example, TGFβ pathway blockade). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CBLB (CBL-B). In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of CISH. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of TIGIT. In some embodiments, temporary alteration of protein expression causes a decrease and/or reduction in the expression of PD-1.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors to, for example, improve TIL transport or motility to tumor sites. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CCRs (chimeric co-stimulatory receptors). In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of chemokine receptors selected from the group consisting of: CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.

In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins. In some embodiments, temporarily altering protein expression causes an increase and/or overexpression of interleukins selected from the group consisting of IL-2, IL-12, IL-15, and/or IL-21.

In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of NOTCH 1/2 ICD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of VHL. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of CD44. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of PIK3CD. In some embodiments, temporary alteration of protein expression causes an increase and/or overexpression of SOCS1.

In some implementations, temporarily altering protein expression causes a decrease and/or reduction in the expression of cAMP protein kinase A (PKA).

In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of two molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one molecule selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of the following: PD-1, CTLA-4, LAG-3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and one of the following: CTLA-4, LAG3, CISH, CBL-B, TIM3, TIGIT, and combinations thereof. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CTLA-4. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of PD-1 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIM-3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CTLA-4 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of LAG3 and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of CISH and TIGIT. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and PD-1. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and LAG3. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CISH. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and CBL-B. In some embodiments, temporarily altering protein expression causes a decrease and/or reduction in the expression of TIM3 and TIGIT.

In some implementations, adhesion molecules selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof are inserted into a first TIL population, a second TIL population, or a collected TIL population via gamma retrovirus or lentivirus methods (e.g., increased expression of adhesion molecules).

In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, temporarily altering protein expression results in a decrease and/or reduction in the expression of molecules selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBL-B, and combinations thereof, and an increase and/or enhancement in the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, expression is reduced by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some implementations, expression is reduced by at least approximately 90%. In some implementations, expression is reduced by at least approximately 95%. In some implementations, expression is reduced by at least approximately 99%.

In some embodiments, expression is increased by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some implementations, expression increases by at least about 90%. In some implementations, expression increases by at least about 95%. In some implementations, expression increases by at least about 99%.

In some embodiments, the temporary alteration of protein expression is induced by treating the TIL with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in the TIL. In some embodiments, intracellular delivery of transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression is performed using a microfluidic platform without SQZ vectors. Such methods have demonstrated the ability to deliver proteins comprising transcription factors to a variety of primary human cell types, including T cells, as described in U.S. Patent Application Publications Nos. US2019/0093073A1, US2018/0201889A1, and US2019/0017072A1, the disclosures of which are each incorporated herein by reference. Such methods can be used in this invention to expose a TIL population to transcription factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression provide an increase in the expression of tumor antigens and/or the number of tumor antigen-specific T cells in the TIL population, thereby leading to TIL population reprogramming and increased therapeutic efficacy of the reprogrammed TIL population compared to the non-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in effector T cells and/or central memory T cell subsets relative to the initial or previous TIL population (i.e., prior to reprogramming), as described herein.

In some embodiments, the transcription factor (TF) includes (but is not limited to) TCF-1, NOTCH 1/2 ICD, and/or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1/2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered with an induced pluripotent stem cell culture (iPSC), such as a commercially available KNOCKOUT serum alternative (Gibco/ThermoFisher), to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered with an iPSC mixture to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is not administered with an iPSC mixture. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some implementations, reprogramming causes a percentage increase in TSCM of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% of TSCM.

In some embodiments, the method of temporarily altering protein expression as described above may be combined with a method of genetically modifying TIL populations, including a step of stabilizing the incorporation of genes for producing one or more proteins. In some embodiments, the method includes a step of genetically modifying TIL populations. In some embodiments, the method includes genetically modifying a first TIL population, a second TIL population, and/or a third TIL population. In some embodiments, the method of genetically modifying TIL populations includes a retroviral transduction step. In some embodiments, the method of genetically modifying TIL populations includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in: Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71; and U.S. Patent No. 6,627,442, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a gamma-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include transposases provided as DNA expression vectors or as expressible RNA or proteins, such that long-term expression of the transposase does not occur in transgenic cells, for example, transposases provided as mRNA (e.g., mRNA containing a cap and a polyadenylated tail). Suitable transposon-mediated gene transfer systems, including salmon-like Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzyme activity, are described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of which are each incorporated herein by reference.

In some implementations, temporary alteration of protein expression in the TIL is induced by small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, which is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where it interferes with the expression of specific genes having complementary nucleotide sequences.

In some implementations, the temporary alteration of protein expression is a reduction in expression. In some implementations, the temporary alteration of protein expression in the TIL is induced by self-delivered RNA interference (sdRNA), which is a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or _-OCH3 ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand bound to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence. sdRNA is a covalently and hydrophobically modified RNAi compound that does not require a delivery medium to enter the cell. sdRNA is typically an asymmetrically modified nucleic acid molecule with a very small double-stranded region. sdRNA molecules typically contain single-stranded and double-stranded regions, and various chemical modifications can be incorporated into these regions. Furthermore, as described herein, sdRNA molecules can be linked to hydrophobic conjugates such as conventional and higher sterol molecules. sdRNAs and related methods for their preparation have been extensively described, for example, in U.S. Patent Application Publications US2016/0304873 A1, US2019/0211337A1, US2009/0131360A1, and US2019/0048341A1, and U.S. Patents 10,633,654 and 10,913,948B2, the disclosures of which are each incorporated herein by reference. To optimize sdRNA structure, chemical properties, target location, sequence preference, etc., an algorithm has been developed and used for sdRNA efficacy prediction. Based on these analyses, a functional sdRNA sequence is generally defined as one whose expression is reduced by more than 70% at a concentration of 1 μM, with a probability exceeding 40%.

Double-stranded DNA (dsRNA) can generally be used to define any molecule containing a pair of complementary RNA strands, typically sense (passenger) and antisense (guide) strands, and may include single-stranded overhang regions. Unlike siRNA, the term dsRNA generally refers to a precursor molecule comprising the sequence of an siRNA molecule, which is released from a larger dsRNA molecule by the action of a lysin system (including Dicer).

In some implementations, the method includes temporarily altering protein expression in a population of TILs (including TILs modified to express CCR), including using self-delivered RNA interference (sdRNA), which is, for example, a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or -OCH ₃ ), comprising a 20-nucleotide antisense (guide) strand and a 13- to 15-base sense (passenger) strand that binds to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Methods using siRNA and sdRNA have been described in the following: Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248; Byrne et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93, the disclosures of which are incorporated herein by reference. In some embodiments, siRNA delivery is accomplished using electroporation or cell membrane disruption (e.g., squeezing or SQZ). In some embodiments, delivery of siRNA or sdRNA to a TIL population does not require electroporation, SQZ, or other methods; the TIL population is actually exposed to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, the method includes delivery of siRNA or sdRNA to a TIL population, which involves exposing the TIL population to siRNA or sdRNA at a concentration of 1 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 10 μM/10,000 TILs in culture medium for a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration of 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days. In some embodiments, delivery of siRNA or sdRNA to a TIL population is accomplished by exposing the TIL population to siRNA or sdRNA at a concentration between 0.1 μM/10,000 TILs and 50 μM/10,000 TILs in the culture medium over a period of 1 to 3 days, wherein the exposure to siRNA or sdRNA is performed two, three, four, or five times by adding fresh siRNA or sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. Patent Application Publications Nos. US 2011/0039914A1, US 2013/0131141A1 and US 2013/0131142A1, and U.S. Patent No. 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA is inserted into the TIL population during manufacturing. In some embodiments, the sdRNA encodes RNA that interferes with the following: NOTCH 1/2 ICD, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and/or CBL-B. In some embodiments, the expression reduction is determined based on the percentage of gene silencing assessed, for example, by flow cytometry and/or qPCR. In some embodiments, the expression reduction is approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

Self-deliverable RNAi technology based on chemically modified siRNA can be used in the methods of this invention to successfully deliver sdRNA to TILs as described herein. The combination of backbone modification with asymmetric siRNA structures and hydrophobic ligands (see, for example, Ligtenberg et al., Mol. Therapy, 2018, 26, 1482-93 and U.S. Patent Application Publication No. 2016/0304873A1, the disclosure of which is incorporated herein by reference) allows sdRNA to permeate cultured mammalian cells simply by adding it to the culture medium, utilizing the nuclease stability of sdRNA without the need for additional formulations and methods. This stability allows for the support of constant levels of RNAi-mediated reduction in target gene activity simply by maintaining an effective concentration of sdRNA in the culture medium. Although not bound by theory, backbone stabilization of sdRNA provides extended reductions in gene expression that can persist for months in non-dividing cells.

In some embodiments, over 95% TIL transfection efficiency and target expression reduction occur through various specific siRNAs or sdRNAs. In some embodiments, siRNAs or sdRNAs containing several unmodified ribose residues undergo fully modified sequence substitutions to increase the potency and/or lifetime of the RNAi effect. In some embodiments, the expression reduction effect lasts for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or longer. In some embodiments, the expression reduction effect decreases after siRNA or sdRNA treatment of TILs for 10 days or longer. In some embodiments, target expression is reduced by more than 70%. In some embodiments, target expression in TILs is reduced by more than 70%. In some embodiments, expression reduction in the PD-1/PD-L1 pathway allows TILs to exhibit a more potent in vivo effect, which in some embodiments is due to avoiding the inhibitory effects of the PD-1/PD-L1 pathway. In some embodiments, TIL proliferation is increased due to reduced PD-1 expression by siRNA or sdRNA.

In some embodiments, the sdRNA sequence used in this invention exhibits a 70% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 75% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 80% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits an 85% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 90% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 95% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a 99% reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM to about 4 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits a reduction in target gene expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced target gene expression when delivered at a concentration of about 4.0 μM.

In some embodiments, the siRNA or sdRNA oligonucleotide agent comprises one or more modifications to increase the stability and/or efficacy of the therapeutic agent and to enable efficient delivery of the oligonucleotide to the cells or tissues to be treated. Such modifications may include 2'-O-methyl modifications, 2'-O-fluorine modifications, dithiophosphate modifications, 2'F-modified nucleotides, 2'-O-methyl-modified and/or 2'-deoxynucleotides. In some embodiments, the oligonucleotide is modified to include one or more hydrophobic modifications, including, for example, sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and/or phenyl. In some embodiments, the chemically modified nucleotide is a combination of thiophosphate, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and thiophosphate. In some embodiments, the sugar may be modified, and the modified sugar may include (but is not limited to) D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halogen (including 2'-fluorine), T-methoxyethoxy, 2'-allyloxy ( _-OCH₂CH = _CH₂ ), 2'-propynyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano. In some embodiments, the sugar moiety may be a hexose and incorporated into an oligonucleotide, such as Augustyns et al., Nucl. Acids. Res. 1992, 18, 4711, the disclosure of which is incorporated herein by reference.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is double-stranded along its entire length, i.e., without a dangling single-stranded sequence at either end of the molecule, i.e., a blunt end. In some embodiments, individual nucleic acid molecules may have different lengths. In other words, the double-stranded siRNA or sdRNA oligonucleotide of the present invention is not double-stranded along its entire length. For example, when using two separate nucleic acid molecules, one of the molecules, such as the first molecule containing an antisense sequence, may be longer than the second molecule with which it hybridizes (leaving a portion of the molecule single-stranded). In some embodiments, when using a single nucleic acid molecule, a portion of the molecule at either end may remain single-stranded.

In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention contain mismatches and/or loops or protrusions, but are double-stranded for at least about 70% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded for at least about 80% of the oligonucleotide length. In other embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 90%-95% of the oligonucleotide length. In some embodiments, the double-stranded siRNA or sdRNA oligonucleotides of the present invention are double-stranded for at least about 96%-98% of the oligonucleotide length. In some embodiments, the double-stranded oligonucleotides of the present invention contain at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

In some embodiments, siRNA or sdRNA oligonucleotides may be substantially protected against nucleases, for example, by modifying the 3' or 5' bonds, as described in U.S. Patent No. 5,849,902, the disclosure of which is incorporated herein by reference. For example, oligonucleotides may be made resistant by incorporating a “blocking group.” As used herein, the term “blocking group” refers to a substituent (e.g., other than an OH group) that can be linked to the oligonucleotide or nucleoside as a protecting or coupling group for synthesis (e.g., FITC, propyl ( _CH₂ - _CH₂ - _CH₃ ), diol (-O- _CH₂ - ^CH₂ _- O-) phosphate ( _PO₃²⁻ ), hydrogen phosphonate, or aminophosphite). A “blocking group” may also include a “terminal blocking group” or “exonuclease blocking group” that protects the 5' and 3' ends of the oligonucleotide, comprising modified nucleotide and non-nucleotide exonuclease resistant structures.

In some implementations, at least a portion of the continuous polynucleotides within the siRNA or sdRNA are linked by substituents, such as phosphate thioesters.

In some embodiments, chemical modification may result in at least 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 13% Cellular uptake of siRNA or sdRNA is enhanced at 0%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, multiple C and U residues contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U may contain hydrophobic modifications. In some embodiments, all C and U contain hydrophobic modifications.

In some embodiments, siRNA or sdRNA molecules exhibit enhanced endosome release via the incorporation of a protonable amine. In some embodiments, the protonable amine is incorporated into the sense strand (the molecular portion discarded after RISC loading). In some embodiments, the siRNA or sdRNA compounds of the present invention comprise an asymmetric compound comprising a double-stranded region (required for efficient RISC entry, 10-15 bases long) and a single-stranded region 4-12 nucleotides long; a double-stranded region having 13 nucleotides. In some embodiments, a single-stranded region of 6 nucleotides is employed. In some embodiments, the single-stranded region of the siRNA or sdRNA comprises 2-12 phosphate thioester nucleotide links (referred to as phosphate thioester modification). In some embodiments, 6-8 phosphate thioester nucleotide links are employed. In some embodiments, the siRNA or sdRNA compounds of the present invention further include unique chemical modification patterns that provide stability and compatibility with RISC entry. For example, the guide strand can also be modified by any chemical modification that demonstrates stability without interfering with RISC entry. In some implementations, the chemical modification pattern in the guide chain includes 2'F modification of most C and U nucleotides and phosphorylation at the 5' end.

In some embodiments, at least 30% of the nucleotides in the siRNA or sdRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6 3%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides are modified. In some embodiments, 100% of the nucleotides in the siRNA or sdRNA are modified.

In some embodiments, the siRNA or sdRNA molecule has a very small double-stranded region. In some embodiments, the double-stranded region of the molecule is between 8 and 15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides in length. There may be 100% complementarity between the guide strand and the passenger strand, or one or more mismatches may exist between the guide strand and the passenger strand. In some embodiments, the molecule has a blunt end or a dangling structure with one nucleotide at one end. The single-stranded region of the molecule is between 4 and 12 nucleotides in length in some embodiments. In some embodiments, the single-stranded region may be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In some embodiments, the single-stranded region may also be less than 4 nucleotides or greater than 12 nucleotides in length. In some embodiments, the single-stranded region is 6 or 7 nucleotides in length.

In some embodiments, the siRNA or sdRNA molecules exhibit increased stability. In some cases, the chemically modified siRNA or sdRNA molecules have a half-life in culture medium longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours, including any intermediate values. In some embodiments, the siRNA or sdRNA has a half-life in culture medium exceeding 12 hours.

In some embodiments, siRNA or sdRNA is optimized to increase efficacy and/or reduce toxicity. In some embodiments, the nucleotide length of the guide strand and/or passenger strand and/or the number of phosphate thioester modifications in the guide strand and/or passenger strand can affect the efficacy of the RNA molecule in some respects, while replacing 2'-fluorine (2'F) modifications with 2'-O-methyl (2'OMe) modifications can affect the toxicity of the molecule in some respects. In some embodiments, reducing the 2'F content of the molecule is expected to reduce the toxicity of the molecule. In some embodiments, the number of phosphate thioester modifications in the RNA molecule can affect the efficiency of molecule uptake into cells, for example, passive uptake of molecules into cells. In some embodiments, siRNA or sdRNA does not have 2'F modifications, but is characterized by equivalent efficacy in cellular uptake and tissue penetration.

In some embodiments, the guide chain is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate ester modifications. For example, the guide chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate ester-modified nucleotides. The guide chain may contain one or more modifications that confer increased stability without interfering with RISC entry. Phosphate-ester-modified nucleotides, such as thiophosphate-modified nucleotides, may be at the 3' end, 5' end, or throughout the entire guide chain. In some embodiments, the 3' end 10 nucleotides of the guide chain contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 thiophosphate-modified nucleotides. The guide chain may also contain 2'F and/or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at position 1 in the guide chain (the nucleotide at the 5' end of the guide chain) is modified with 2'OMe and/or phosphorylated. C and U nucleotides within the guide chain may be modified with 2'F. For example, the C and U nucleotides at positions 2-10 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'F. The C and U nucleotides within the guide chain may also be modified with 2'OMe. For example, the C and U nucleotides at positions 11-18 (or corresponding positions in guide chains of different lengths) of the 19-nucleotide guide chain may be modified with 2'OMe. In some embodiments, the nucleotides at the 3' end of the guide chain are unmodified. In some embodiments, most of the C and U nucleotides within the guide chain are modified with 2'F, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, and the 5' end of the guide chain is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with 2'OMe, the 5' end of the guide chain is phosphorylated, and the C or U at positions 2-10 are modified with 2'F.

Self-deliverable RNAi technology provides a method for directly transfecting cells with an RNAi agent (whether siRNA, sdRNA, or other RNAi agents) without additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity, and ease of use are characteristics of the compositions and methods, which offer significant functional advantages over conventional siRNA-based techniques, and therefore the sdRNA method is employed in several embodiments of the method for reducing target gene expression in TILs of the present invention. The sdRNA method allows for the direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues in vitro and in vivo. The sdRNAs described in some embodiments of the invention herein are available from Advirna LLC, Worcester, MA, USA.

siRNA and sdRNA can be formed by hydrophobically modified siRNA-antisense oligonucleotide hybridization and are disclosed, for example, in Byrne et al., J. Ocular Pharmacol. Therapeut., 2013, 29, 855-864, the contents of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA oligonucleotides may be delivered to the TILs described herein using sterile electroporation. In some embodiments, the method includes sterile electroporation of TIL populations to deliver siRNA or sdRNA oligonucleotides.

In some embodiments, the oligonucleotide may be delivered to cells in combination with a transmembrane delivery system. In some embodiments, this transmembrane delivery system includes lipids, viral vectors, etc. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that does not require any delivery agent. In some embodiments, the method includes using a transmembrane delivery system to deliver siRNA or sdRNA oligonucleotides to a TIL population.

The oligonucleotides and oligonucleotide compositions are contacted (e.g., brought into contact, also referred to herein as being administered or delivered to) and taken up, including passively via the TIL. sdRNA may be added to the TIL as described herein at the following times: during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D), after step D and before collection in step E, during or after collection in step F, before or during final formulation and/or transfer to the infusion bag in step F, and before any optional cryopreservation step in step F. Furthermore, sdRNA may be added after thawing from any cryopreservation step in step F. In some embodiments, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TIL and other agents at concentrations selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM. In some implementations, one or more sdRNAs targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to a cell culture medium containing TILs and other agents in amounts selected from the group consisting of: 0.1 μM sdRNA/10,000 TILs/100 μL of medium, 0.5 μM sdRNA/10,000 TILs/100 μL of medium, 0.75 μM sdRNA/10,000 TILs/100 μL of medium. IL/100μL medium, 1μM sdRNA/10,000 TILs/100μL medium, 1.25μM sdRNA/10,000 TILs/100μL medium, 1.5μM sdRNA/10,000 TILs/100μL medium, 2μM sdRNA/10,000 TILs/100μL medium, 5μM sdRNA/10,000 TILs/100μL medium, or 10μM sdRNA/10,000 TILs/100μL medium. In some implementations, one or more sdRNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBL-B) may be added to TIL cultures twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the pre-REP or REP phase.

The oligonucleotide compositions of the present invention, including siRNA or sdRNA, can be contacted with TILs as described herein during an amplification process, for example, by dissolving high concentrations of siRNA or sdRNA in a cell culture medium and allowing sufficient time for passive uptake. In some embodiments, the method of the present invention includes contacting a TIL population with the oligonucleotide composition as described herein. In some embodiments, the method includes dissolving an oligonucleotide (e.g., siRNA or sdRNA) in a cell culture medium and contacting the cell culture medium with the TIL population. TILs may be a first, second, and/or third population as described herein.

In some embodiments, delivery of oligonucleotides into cells can be enhanced by suitable methods known in the art, including calcium phosphate, DMSO, glycerol or polydextrose, electroporation, or by transfection, for example using cationic, anionic or neutral lipid compositions or liposomes, using methods known in the art, such as those described in: U.S. Patent Nos. 4,897,355; 5,459,127; 5,631,237; 5,955,365; 5,976,567; 10,087,464; and 10,155,945; and Bergan et al., Nucl. Acids Res. 1993, 21,3567, the disclosures of which are each incorporated herein by reference.

In some embodiments, more than one siRNA or sdRNA is used to reduce the expression of a target gene. In some embodiments, one or more of PD-1, TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH that target siRNA or sdRNA are used together. In some embodiments, PD-1 siRNA or sdRNA is used with one or more of TIM-3, CBL-B, LAG3, CTLA-4, TIGIT, and/or CISH to reduce the expression of more than one gene target. In some embodiments, LAG3 siRNA or sdRNA is used in combination with CISH that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, TIGIT siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CISH siRNA or sdRNA is used in combination with PD-1 that targets siRNA or sdRNA to reduce the expression of two genes. In some embodiments, CTLA-4 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, LAG-3 siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, CBL-B siRNA or sdRNA is used in combination with PD-1 targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, the siRNA or sdRNA targeting one or more of PD-1, TIM-3, CBL-B, LAG3, and/or CISH described herein is available from Advirna LLC, Worcester, Massachusetts, USA, or from several other suppliers.

In some embodiments, siRNA or sdRNA targets genes selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets genes selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof. In some embodiments, the siRNA or sdRNA targets genes selected from PD-1, LAG-3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from PD-1 and one of the following: LAG3, CISH, CBL-B, TIM3, and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets PD-1, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets LAG3, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets CISH, and another siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets PD-1. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets TIM3, and another siRNA or sdRNA targets CBL-B.

As discussed herein, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been genetically modified by gene editing to enhance their therapeutic efficacy. Embodiments of the present invention cover gene editing of TIL populations via nucleotide insertion (RNA or DNA) to promote and inhibit the expression of one or more proteins, and combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into therapeutic populations, wherein the methods include gene editing of the TILs. Several gene editing techniques exist that can be used to genetically modify TIL populations, said gene editing techniques being suitable for use according to the present invention. Such methods include those described below as well as viral and transposon methods described elsewhere herein. In some embodiments, methods for genetically modifying TILs, MILs, or PBLs to express CCRs may further include modifications to inhibit gene expression via stable gene knockout or temporary gene knockdown of such genes.

In some embodiments, the method includes methods for genetically modifying TIL populations in a first, second, and/or third population as described herein. In some embodiments, the method for genetically modifying TIL populations includes a step of stabilizing a gene incorporated for producing or repressing (e.g., silencing) one or more proteins. In some embodiments, the method for genetically modifying TIL populations includes an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are each incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein at least two of the at least three pulses are different from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator-controlled, independently programmed DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first group is different from the second pulse interval of two of the at least three pulses in the second group. In some embodiments, the electroporation method is a pulsed electroporation method, which includes the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, including the step of applying a series of at least three DC electrical pulses to the TIL, the field strength being equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two pulses in the first group of at least three pulses is different from the second pulse interval of the two pulses in the second group of at least three pulses, such that the induced pores persist for a relatively long period of time, and such that the survival rate of the TIL is maintained. In some embodiments, the method of genetically modifying the TIL population includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are each incorporated herein by reference. In some embodiments, methods for genetically modifying TIL populations include a liposome transfection step. Liposome transfection methods, such as those using cationic lipids N-[1-(2,3-diolenoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water in a 1:1 (w/w) liposome formulation, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patents 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population includes a transfection step using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of which are each incorporated herein by reference. The TIL may be a first TIL population, a second TIL population, and/or a third TIL population as described herein.

According to one embodiment, gene editing methods may include the use of programmable nucleases that mediate the generation of double-strand or single-strand breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on recognizing specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand breaks in the DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homologous directed repair (HDR). Thus, the repair of the breaks can lead to the introduction of insertion/deletion mutations that disrupt (e.g., silence, repress, or enhance) the product of the target gene.

The main classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems (e.g., Cas9) target specific DNA sequences through short RNA guide molecules that directly pair with the target DNA and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that can be used in the TIL amplification method according to the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to one embodiment, the method for amplifying TILs into a therapeutic population can be performed according to any embodiment of the method described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein the method further includes gene editing of at least a portion of the TIL by one or more of the CRISPR method, the TALE method, or the ZFN method to produce TILs that provide enhanced therapeutic effects. According to one embodiment, the improved therapeutic effect of gene-edited TILs can be evaluated by in vitro comparison of gene-edited TILs with unmodified TILs, for example by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs. In some implementations, the method includes gene editing of TIL populations using CRISPR, TALE, and/or ZFN methods.

In some embodiments of the invention, electroporation is used to deliver gene editing systems such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow cytometry electroporation system. An example of a suitable flow cytometry electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. Several alternative commercially available electroporation instruments may be suitable for the invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed, sterile system together with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system together with the remainder of the TIL amplification method.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs using a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf1). According to a particular embodiment, the CRISPR method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the CRISPR method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

CRISPR stands for clustered regularly spaced short palindromic repeats. The method of gene editing using the CRISPR system is also referred to herein as the CRISPR method. There are three types of incorporating RNA and Cas proteins and CRISPR systems that can be used according to the present invention: type I, II, and III. Type II CRISPR (using Cas9 as an example) is one of the most fully characterized systems.

CRISPR technology is adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins (including Cas9) to prevent attacks from viruses and other foreign organisms by cleaving and destroying the DNA of invading foreign organisms. CRISPR is a DNA-specialized region with two unique characteristics: nucleotide repeat sequences and spacers. The nucleotide repeat sequences are distributed throughout the CRISPR region, with short foreign DNA segments (spacers) interspersed within the repeat sequences. In the type II CRISPR/Cas system, the spacers are integrated into the CRISPR genomic locus and transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNA (tracrRNA) and guide the Cas proteins to perform sequence-specific cleavage and silence pathogenic DNA. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved protospacer adjacent motif (PAM) sequence containing a dinucleotide upstream of the crRNA-binding region. Thus, the CRISPR/Cas system can retarget to cleave almost any DNA sequence by redesigning the crRNA. The crRNA and tracrRNA in the native system can be simplified to a single guide RNA (sgRNA) of approximately 100 nucleotides for genetic engineering. The CRISPR/Cas system can be directly introduced into human cells by co-delivering plasmids expressing the Cas9 endonuclease and the essential crRNA component. Different Cas protein variants can be used to reduce targeting limitations (e.g., heterologous homologs of Cas9, such as Cpf1).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via CRISPR methods include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. , CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences using CRISPR methods and applicable to embodiments of the invention are described in U.S. Patents 8,697,359, 8,993,233, 8,795,965, 8,771,945, 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 8,871,445, 8,906,616, and 8,895,308, the disclosures of which are each incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are available from companies such as GenScript.

In some implementations, genetic modification of the TIL population as described herein can be performed using a CRISPR/Cpf1 system as described in U.S. Patent No. US9790490, the disclosure of which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publications Nos. US 2020/0299644A1 and US 2020/0121719A1, and U.S. Patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a TALE method. According to a particular embodiment, the TALE method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the TALE method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for transcription activator-like effector protein, which includes transcription activator-like effector nucleases (TALENs). The method of gene editing using the TALE system is also referred to herein as the TALE approach. TALEs are naturally occurring proteins from the plant pathogenic bacterium *Xanthomonas* and contain a DNA-binding domain consisting of a series of repeating domains of 33–35 amino acids, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeat sequences are linked together to recognize consecutive DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of a TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to prepare a targeted TALE nuclease. To induce site-specific mutations, two individual TALEN arms, separated by a 14–20 base-pair spacer region, pull FokI monomers together to dimerize and generate targeted double-strand breaks.

Several large, systematic studies utilizing various assembly methods indicate that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuti cals (Lexington, Kentucky, KY, USA), and Life Technolo gies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this invention are described in U.S. Patent Application Publications Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1, and US 2016/0120906 A1, the disclosures of which are each incorporated herein by reference.

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, and TNFRSF10A. CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL 6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of a target gene sequence via the TALE method and which can be used according to embodiments of the invention are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

Methods for amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein or as described in U.S. Patent Application Publications US2020/0299644 A1 and US2020/0121719 A1, and U.S. Patent No. 10,925,900 (the disclosure of which is incorporated herein by reference), wherein said methods further include gene editing of at least a portion of the TILs by a zinc finger or zinc finger nuclease method. According to a particular embodiment, the zinc finger method is used during the TIL amplification process to induce silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the zinc finger method is used during the TIL amplification process to induce enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Individual zinc fingers, exhibiting a conserved ββα conformation, contain approximately 30 amino acids. Several amino acids on the α-helix surface typically contact the 3 bp in the main groove of the DNA at varying levels of selectivity. Zinc fingers possess two protein domains. The first domain is a DNA-binding domain, which includes eukaryotic transcription factors and contains the zinc finger. The second domain is a nuclease domain, which includes the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

Individual zinc finger (ZFN) DNA-binding domains typically contain between three and six individual zinc finger repeats and can each recognize between nine and 18 base pairs. If the zinc finger domains are specific for their intended target sites, even a pair of three-finger ZFNs recognizing a total of 18 base pairs can theoretically target a single locus in the mammalian genome. One method for generating novel zinc finger arrays is to combine smaller zinc finger “modules” with known specificity. The most common module assembly process involves combining three separate zinc fingers, each recognizing a 3-base-pair DNA sequence, to produce a three-finger array recognizing a 9-base-pair target site. Alternatively, selection-based methods, such as oligopool engineering (OPEN), can be used to select new zinc finger arrays from a randomly grouped library that considers context-dependent interactions between neighboring fingers. Engineered zinc fingers are available from Sangamo Biosciences (Richmond, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA).

Non-restricted examples of genes that can be silenced or repressed by permanent gene editing of TILs via the zinc finger method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, and C. ASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6 R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-restricted examples of genes that can be enhanced by permanent gene editing of TILs via the zinc finger method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which can be used according to embodiments of the invention are described in U.S. Patents 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, each of which is incorporated herein by reference.

Examples of systems, methods, and compositions for altering the expression of target gene sequences via zinc finger methods and which may be used according to other embodiments of the invention are described in Beane et al., Mol. Therapy, 2015, 23, 1380-1390, the disclosure of which is incorporated herein by reference.

In some embodiments, TILs are optionally genetically engineered to include other functionalities, including (but not limited to) high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). In some embodiments, the method includes genetically engineering a population of TILs to include high-affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19). Suitablely, the TIL population may be a first, second, and/or third population as described herein.

D. Closed systems used in TIL manufacturing

This invention provides a closed system for use during TIL (Total Intake Liquid) culture. Such a closed system allows for the prevention and/or reduction of microbial contamination, allows for the use of fewer culture flasks, and allows for lower costs. In some embodiments, the closed system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

A sterile connection device (STCD) creates a sterile weld between two compatible tubes. This procedure allows for the aseptic connection of multiple containers and tube diameters. In some embodiments, the closure system includes a Luer lock and a heat-sealing system as described in the examples. In some embodiments, the closure system is introduced under aseptic conditions via a syringe to maintain the sterility and closure properties of the system. In some embodiments, a closure system as described in the examples is used. In some embodiments, the TIL is formulated into a final product formulation container according to the method described in the examples herein.

In some embodiments, the closed system uses a single container from the time the tumor fragment is obtained until it is ready for TIL administration to the patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G container, and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. The closed system or closed TIL cell culture system is characterized by tightly sealing the system from the outside to create a closed environment free from bacterial, fungal, and/or other microbial contamination once the tumor sample and/or tumor fragment has been added.

In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

The closed system allows TILs to grow in the absence of microbial contamination and/or with significantly reduced microbial contamination.

Furthermore, the pH, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment each change with cell culture. Therefore, even if the culture medium suitable for cell culture is circulated, the closed environment still needs to be continuously maintained to provide the optimal environment for TIL proliferation. For this purpose, it is desirable to monitor the physical factors of pH, carbon dioxide partial pressure, and oxygen partial pressure within the culture medium of the closed environment using sensors. These signals are used to control a gas exchanger installed at the inlet of the culture environment, and the gas partial pressure of the closed environment is adjusted in real time according to changes in the culture medium to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system incorporating a gas exchanger equipped with monitoring devices for measuring the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment at the inlet, and optimizing the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring devices.

In some implementations, the pressure within the closed environment is controlled continuously or intermittently. That is, the pressure within the closed environment can be varied by means of, for example, a pressure maintaining device, thereby ensuring that the space is suitable for TIL growth under positive pressure or promoting exudation of fluid and thus promoting cell proliferation under negative pressure. Furthermore, by intermittently applying negative pressure, it is possible to uniformly and effectively displace the circulating fluid within the closed environment by temporarily reducing its volume.

In some implementations, the optimal culture components for TIL proliferation may be replaced or added, and factors including, for example, IL-2 and/or OKT3, and combinations thereof may be added.

E.TIL's optional cryopreservation

Optionally, the primary TIL population (e.g., a second TIL population) or an amplified TIL population (e.g., a third TIL population) can be cryopreserved. In some embodiments, therapeutic TIL populations are cryopreserved. In some embodiments, cryopreservation occurs on TILs collected after a second amplification. In some embodiments, TILs are cryopreserved in exemplary step F of Figures 1 and/or 8 (particularly, e.g., Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs are cryopreserved in infusion bags. In some embodiments, TILs are cryopreserved before being placed in infusion bags. In some embodiments, TILs are cryopreserved without being placed in infusion bags. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is typically accomplished by placing a population of TILs in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are then placed in cryopreservation vials and stored at -80°C for 24 hours, with optional transfer to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some embodiments, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLifeSolutions). In some embodiments, the TIL population is cryopreserved using cryopreservation medium containing dimethyl sulfoxide (DMSO). In some embodiments, the TIL population is cryopreserved using a 1:1 (vol:vol) ratio of CS10 to cell culture medium. In some embodiments, the TIL population is cryopreserved using approximately a 1:1 (vol:vol) ratio of CS10 to cell culture medium (also containing additional IL-2).

As discussed above and illustrated in steps A through E in Figures 1 and/or 8 (especially, for example, Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P), cryopreservation can occur at multiple points in the TIL amplification process. In some embodiments, the expanded TIL population after the first amplification (as provided, for example, according to step B) or after one or more second amplifications according to step D of Figure 1 or Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be cryopreserved. Cryopreservation is typically accomplished by placing the TIL population in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution are placed in cryopreservation vials and stored at -80°C for 24 hours, wherein optionally they are transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, TILs are cryopreserved according to the method provided in Example 6.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, the thawed TILs can be counted and their viability assessed as known in the art.

In some cases, the TIL populations of step B in Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) can be immediately cryopreserved using the schemes discussed below. Alternatively, the main TIL population may undergo steps C and D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), and then cryopreservation following step D from Figure 1 or Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). Similarly, in cases where genetically modified TILs will be used in a therapy, the TIL population from step B or step D of Figure 1 or Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) may undergo genetic modification for appropriate treatment.

F. Phenotypic characteristics of amplified TILs

In some embodiments, the expression of TILs after amplification is analyzed using multiple phenotypic markers, including those described herein and in the examples. In some embodiments, the expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characterization of TILs is analyzed after the first amplification in step B of FIG1 or FIG8 (particularly, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transition in step C from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed during the transformation from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) and after cryopreservation. In some implementations, the phenotypic characteristics of TIL are analyzed after the second amplification in step D from Figure 1 or Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, the phenotypic characteristics of TIL are analyzed after two or more amplifications from step D of FIG1 or FIG8 (in particular, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P).

In some implementations, the marker is selected from the group consisting of CD8 and CD28. In some implementations, the expression of CD8 is examined. In some implementations, the expression of CD28 is examined. In some implementations, compared to other processes (e.g., the Gen 3 process provided in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)), compared to the Gen 3 process provided in Figure 8A and/or Figure 8B, the expression of CD28 is examined. The 2A process provided in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P) produces a higher expression of CD8 and/or CD28 on the TIL according to the process of the present invention. In some implementations, the expression of CD8 on the TIL produced by the process according to the invention is higher than that of other processes (e.g., the Gen 3 process provided in, for example, FIG8 (especially, for example, FIG8B)) and the 2A process provided in, for example, FIG8 (especially, for example, FIG8A and/or FIG8B and/or FIG8C and/or FIG8D and/or FIG8E and/or FIG8F and/or FIG8G and/or FIG8H and/or FIG8I and/or FIG8J and/or FIG8K and/or FIG8L and/or FIG8M and/or FIG8N and/or FIG8O and/or FIG8P). In some embodiments, the expression of CD28 on the TIL produced by the process according to the invention is higher than that of the 2A process provided, such as, for example, in Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), compared to other processes (e.g., Figure 8A and/or Figure 2A, for example, Figure 8A). In some embodiments, high CD28 expression indicates a younger and more durable TIL phenotype. In some embodiments, the expression of one or more regulatory markers is measured.

In some implementations, during any step of the method for amplifying tumor-infiltrating lymphocytes (TILs) as described herein, no selection of a first TIL population, a second TIL population, a third TIL population, or a collected TIL population is made based on CD8 and/or CD28 expression.

In some embodiments, the percentage of central memory cells in the TIL produced by the method according to the invention is higher than that of process 2A, as provided in, for example, Figure 8 (particularly, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, the memory markers of the central memory cells are selected from the group consisting of CCR7 and CD62L.

In some implementations, the CD4+ and/or CD8+ TIL memory subsets may be divided into different memory subsets. In some implementations, the CD4+ and/or CD8+ TILs contain initial (CD45RA+CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain central memory (CM; CD45RA-CD62L+) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain effect memory (EM; CD45RA-CD62L-) TILs. In some implementations, the CD4+ and/or CD8+ TILs contain RA+ effect memory/effect (TEMRA/TEFF; CD45RA+CD62L+) TILs.

In some embodiments, the TIL expresses one or more markers selected from the group consisting of granzyme B, perforin, and granulosin. In some embodiments, the TIL expresses granzyme B. In some embodiments, the TIL expresses perforin. In some embodiments, the TIL expresses granulosin.

In some embodiments, a cytokine release assay may also be used to evaluate cytokine release from restimulated TILs. In some embodiments, interferon-γ (IFN-γ) secretion from TILs may be evaluated. In some embodiments, IFN-γ secretion is measured by an ELISA assay. In some embodiments, IFN-γ secretion is measured by an ELISA assay after a rapid second amplification step, following step D as provided, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TIL health is measured by IFN-γ (IFN-γ) secretion. In some embodiments, IFN-γ secretion indicates active TILs. In some embodiments, a potency assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the culture medium of TILs stimulated with antibodies against CD3, CD28, and CD137/4-1BB. The IFN-γ level in the culture medium from these stimulated TILs can be determined by measuring IFN-γ release. In some embodiments, for example, Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P), step D in the Gen 3 process provided in the TIL shows increased IFN-γ production compared to step D in the 2A process provided, for example, Figure 8 (especially, for example, Figure 8A), indicating an increased cytotoxic potential of the TIL in step D. In some embodiments, IFN-γ secretion is increased by one, two, three, four, or five times or more. In some embodiments, IFN-γ secretion is increased by one time. In some embodiments, IFN-γ secretion is increased by two times. In some embodiments, IFN-γ secretion is increased by three times. In some embodiments, IFN-γ secretion is increased by four times. In some embodiments, IFN-γ secretion is increased by five times. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ in ex vivo TILs is measured. In some embodiments, IFN-γ in ex vivo TILs is measured, including TILs produced by the methods of the present invention (including, for example, the method of Figure 8B).

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the amount of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times more IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL to about 1000 pg/mL or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some implementations, it is capable of secreting at least 200 pg/mL, at least 250 pg/mL, at least 300 pg/mL, at least 350 pg/mL, at least 400 pg/mL, at least 450 pg/mL, at least 500 pg/mL, at least 550 pg/mL, at least 600 pg/mL, at least 650 pg/mL, at least 700 pg/mL, at least 750 pg/mL, at least 800 pg/mL, at least 850 pg/mL. L, at least 900 pg/mL, at least 950 pg/mL, or at least 1000 pg/mL or more of IFN-γ TILs are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 100 pg/mL/5e5 cells to about 1000 pg/mL/5e5 cells or more of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, it is capable of secreting at least 200 pg/mL/5e5 cells, at least 250 pg/mL/5e5 cells, at least 300 pg/mL/5e5 cells, at least 350 pg/mL/5e5 cells, at least 400 pg/mL/5e5 cells, at least 450 pg/mL/5e5 cells, at least 500 pg/mL/5e5 cells, at least 550 pg/mL/5e5 cells, at least 600 pg/mL/5e5 cells, at least 650 pg/mL/5e5 cells, at least 700 pg/mL/5e5 cells, and at least 750 pg/mL/5e5 cells. TILs of IFN-γ produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P) are TILs produced by at least 800 pg/mL/5e5 cells, at least 850 pg/mL/5e5 cells, at least 900 pg/mL/5e5 cells, at least 950 pg/mL/5e5 cells, or at least 1000 pg/mL/5e5 cells or more. In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 300 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 400 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 600 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 700 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 800 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 900 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 10,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 15,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 20,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 25,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 30,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 35,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 40,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 45,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 50,000 pg/mL/5e5 cells of IFN-γ are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

The diverse antigen receptors of T and B lymphocytes are generated through somatic recombination of a limited but large number of gene regions. These gene regions—V (variable region), D (diversity region), J (binding region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that express and increase the diversity of T cell reservoirs. In some embodiments, TILs obtained by the method of this invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using a method called Gen2 exemplified in Figure 8 (particularly, for example, Figure 8A). In some embodiments, TILs obtained in the first amplification exhibit increased T cell reservoir diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin heavy chain. In some embodiments, the diversity is present in immunoglobulins, specifically in the immunoglobulin light chain. In some embodiments, the diversity is present in T-cell receptors. In some embodiments, the diversity is present in one of the T-cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T-cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T-cell receptor (TCR) α is increased. In some embodiments, the expression of T-cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased. In some embodiments, processes as described herein (e.g., the Gen 3 process) exhibit higher clonal diversity based on the number of unique peptide CDRs within the sample compared to other processes (e.g., the Gen 2 process).

In some embodiments, TIL activation and depletion can be determined by examining one or more biomarkers. In some embodiments, activation and depletion can be determined using multicolor flow cytometry. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: CD3, PD-1, 2B4/CD244, CD8, CD25, BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183, CD69, CD95, CD127, CD103, and/or LAG-3. In some embodiments, biomarker activation and depletion include (but are not limited to) one or more biomarkers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, PD-1, TIGIT, and/or TIM-3. In some embodiments, activation and depletion of markers include (but are not limited to) one or more markers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, CD103+/CD69+, CD103+/CD69-, PD-1, TIGIT, and/or TIM-3. In some embodiments, T cell markers (including activation and depletion markers) may be identified and/or analyzed to examine T cell activation, suppression, or function. In some embodiments, T cell markers may include (but are not limited to) one or more markers selected from the group consisting of: TIGIT, CD3, FoxP3, Tim-3, PD-1, CD103, CTLA-4, LAG-3, BTLA-4, ICOS, Ki67, CD8, CD25, CD45, CD4, and/or CD59.

In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TILs to 300000 pg/ ^10⁶ TILs or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed to be higher than 3000 pg/ ^10⁶ TILs, higher than 5000 pg/ ^10⁶ TILs, higher than 7000 pg/ ^10⁶ TILs, higher than 9000 pg/ ^10⁶ TILs, higher than 11000 pg/ ^10⁶ TILs, higher than 13000 pg/ ^10⁶ TILs, higher than 15000 pg/ ^10⁶ TILs, higher than 17000 pg/ ^10⁶ TILs, higher than 19000 pg/ ^10⁶ TILs, higher than 20000 pg/ ^10⁶ TILs, higher than 40000 pg/ ^10⁶ TILs, higher than 60000 pg/10⁶ TILs, higher than 80000 pg/ ^10⁶ TILs, and higher than 100000 pg/ ^10⁶ TILs. ⁶ TILs, above 120,000 pg/10; ⁶ TILs, above 140,000 pg/10; ⁶ TILs, above 160,000 pg/10; ⁶ TILs, above 180,000 pg/10; ⁶ TILs, above 200,000 pg/10; ⁶ TILs, above 220,000 pg/10; ⁶ TILs, above 240,000 pg/10; ⁶ TILs, above 260,000 pg/10; ⁶ TILs, above 280,000 pg/10; ⁶ TILs, above 300,000 pg/10 TILs with ⁶ or more granzyme B are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 3000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 5000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 7000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 9000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 11,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 13,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 15,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 17,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 19,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 20,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 40,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 60,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 80,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 100,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 260,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/ ^10⁶ TIL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, TILs exhibiting secretion of granzyme B at levels higher than 1000 pg/mL to 300000 pg/mL or more are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some implementations, secretion was observed at levels above 1000 pg/mL, above 2000 pg/mL, above 3000 pg/mL, above 4000 pg/mL, above 5000 pg/mL, above 6000 pg/mL, above 7000 pg/mL, above 8000 pg/mL, above 9000 pg/mL, above 10000 pg/mL, above 20000 pg/mL, above 30000 pg/mL, above 40000 pg/mL, above 50000 pg/mL, and above 60000 pg/mL. TILs with granzyme B concentrations of 0000 pg/mL, above 70000 pg/mL, above 80000 pg/mL, above 90000 pg/mL, above 100000 pg/mL, or more are TILs produced by the amplification method of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 1000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 2000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 3000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 4000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 5000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 6000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 7000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 8000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 9000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 10,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 20,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 30,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 40,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 50,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 60,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 70,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 80,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 90,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting a granzyme B concentration higher than 100,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 120,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 140,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 160,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 180,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 200,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 220,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 240,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of granzyme B above 260,000 pg/mL are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 280,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P). In some embodiments, TILs exhibiting secretion of more than 300,000 pg/mL granzyme B are TILs produced by the amplification methods of the present invention (including, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P).

In some embodiments, the amplification method of the present invention produces an amplified TIL population exhibiting increased in vitro granzyme B secretion compared to a non-amplified TIL population, including, for example, the TILs provided in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased by at least one to fifty times or more compared to a non-amplified TIL population. In some embodiments, IFN-γ secretion is increased by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least thirty, at least forty, at least fifty times or more compared to a non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least doubled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least tripled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least quadrupled compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least five times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least six times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least seven times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is at least eight times increased compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least nine-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least ten-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least twenty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least thirty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least forty-fold compared to the non-amplified TIL population. In some embodiments, the granzyme B secretion of the amplified TIL population of the present invention is increased at least fifty-fold compared to the non-amplified TIL population.

In some embodiments, TILs capable of secreting at least one, two, three, four, or five times lower levels of TNF-α (i.e., TNF-alpha) compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least twice the level of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least three times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least four times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least five times lower levels of TNF-α compared to IFN-γ secretion are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α (i.e., TNF-alpha) are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some embodiments, IFN-γ and granzyme B levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, granzyme B and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P). In some embodiments, IFN-γ, granzyme B, and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention (including, for example, the methods shown in Figures 8A and/or 8B and/or 8C and/or 8D and/or 8E and/or 8F and/or 8G and/or 8H and/or 8I and/or 8J and/or 8K and/or 8L and/or 8M and/or 8N and/or 8O and/or 8P).

In some implementations, phenotypic characteristics are examined after cryopreservation.

G. Other process implementation plans

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days or about 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days or about 1 to 10 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling-up of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein in the second container, the second TIL population from the small-scale culture is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days or approximately 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) transferring and distributing the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 to 7 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for approximately 1 to 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for approximately 1 to 8 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a vertical scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 8 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling up of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 6 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 5 days.

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a subject into multiple tumor fragments; (b) initiating a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and OKT-3, wherein the initiation of the first expansion is performed for about 1 to 7 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3 and exogenous antigen-presenting cells (APCs) to generate a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some implementations, the rapid second amplification step is broken down into multiple steps to achieve a longitudinal scaling up of the culture by: (1) performing rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring the second TIL population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) that is larger than the first container, wherein the second TIL population from the small-scale culture in the second container is cultured in a larger-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve lateral scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, the portion of the second TIL population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In some implementations, the rapid expansion step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second expansion by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 4 to 7 days. In some implementations, the rapid amplification step is broken down into multiple steps to achieve both lateral and vertical scaling of the culture scale by: (1) performing a rapid second amplification by culturing a second TIL population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-g500MCS containers) that are larger than the first container, wherein in each second container, the portion of the second TIL population transferred from the small-scale culture to such a second container is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by contacting a first TIL population with a culture medium that also contains exogenous antigen-presenting cells (APCs), wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the culture medium is supplemented with additional exogenous APC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from a range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 2:1.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, or 2.3:1. 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the initial first amplification is exactly or approximately 1× ^10⁸ , ^1.1 ×10⁸, 1.2× ^10⁸ , 1.3× ^10⁸ , 1.4×10⁸, 1.5× ^10⁸ , 1.6 ^{×10⁸ ,} 1.7× ^10⁸ , 1.8× ^10⁸ , 1.9×10⁸, 2× ^10⁸ , 2.1× ^10⁸ , 2.2× ^10⁸ , 2.3× ^10⁸ , 2.4× ^10⁸ , 2.5× ^10⁸ , 2.6× ^10⁸ , 2.7× ^10⁸ , 2.8× ^10⁸ , 2.9× ^10⁸ , 3× ^10⁸ , 3.1× ^10⁸ , or 3.1×10⁸. ⁸ , 3.2× ^10⁸ , 3.3× ^10⁸ , 3.4× ^10⁸ , or 3.5× ^10⁸ APCs, wherein the number of APCs added in the rapid second amplification is exactly or approximately 3.5× ^10⁸ , 3.6×10⁸, ^3.7 × ^10⁸ , 3.8× ^10⁸ , 3.9× ^10⁸ , 4×10⁸, 4.1× ^10⁸ , ^4.2 × ^10⁸ , 4.3× ^10⁸ , 4.4× ^10⁸ , 4.5×10⁸, 4.6× ^10⁸ , 4.7× ^10⁸ , 4.8× ^10⁸ , 4.9× ^10⁸ , 5× ^10⁸ , 5.1× ^10⁸ , 5.2× ^{10⁸ ,} or 5.3× ^10⁸. 5.4× ^10⁸ , 5.5× ^10⁸ , 5.6×10⁸, 5.7× ^10⁸ , 5.8× ^10⁸ , 5.9× ^10⁸ , 6× ^10⁸ , 6.1× ^10⁸ , 6.2×10⁸, 6.3× ^10⁸ , 6.4× ^10⁸ , 6.5× ^10⁸ , ^6.6 × ^10⁸ , 6.7× ^10⁸ , 6.8× ^10⁸ , 6.9× ^10⁸ , 7× ^10⁸ , ^7.1 × ^10⁸ , 7.2×10⁸, 7.3× ^10⁸ , 7.4× ^10⁸ , 7.5× ^10⁸ , 7.6× ^10⁸ , 7.7× ^10⁸ , ^7.8 × ^10⁸ 7.9× ^10⁸ , 8× ^10⁸ , 8.1×10⁸, 8.2× ^10⁸ , 8.3× ^10⁸ , 8.4× ^10⁸ , 8.5× ^10⁸ , 8.6× ^10⁸ , 8.7×10⁸, 8.8× ^10⁸ , 8.9× ^10⁸ , 9× ^10⁸ , 9.1× ^10⁸ , 9.2× ^10⁸ , 9.3× ^10⁸ , 9.4× ^10⁸ , 9.5× ^10⁸ , ^9.6 × ^10⁸ , 9.7× ^10⁸ , 9.8× ^10⁸ , ^{9.9×10⁸ or} 1× ^10⁹ APCs.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1 × ^10⁸ APCs to exactly or about 3.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 3.5 × ^10⁸ APCs to exactly or about 1 × ^10⁹ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 1.5 × ^10⁸ APCs to exactly or about 3 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4 × ^10⁸ APCs to exactly or about 7.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs added in the preliminary first amplification is selected from the range of exactly or about 2 × ^10⁸ APCs to exactly or about 2.5 × ^10⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4.5 × ^10⁸ APCs to exactly or about 5.5 × ^10⁸ APCs.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or approximately 2.5 × ^10⁸ APCs are added to an initial first amplification, and exactly or approximately 5 × ^10⁸ APCs are added to a rapid second amplification.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein multiple tumor fragments are distributed into multiple separate containers, in each separate container a first TIL population is obtained in step (a), a second TIL population is obtained in step (b), and a third TIL population is obtained in step (c), and therapeutic TIL populations from the multiple containers in step (c) are combined to produce a collected TIL population from step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of tumors are uniformly distributed into a plurality of separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise at least two separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to twenty separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to fifteen separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to ten separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of separate containers comprise two to five separate containers.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the plurality of separate containers comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first amplification of a first TIL population in step (b) is performed in each container, and a rapid second amplification of a second TIL population generated from such a first TIL population is performed in the same container as in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each of the separate containers includes a first breathable surface area.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein multiple tumor fragments are distributed in a single container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a single container includes a first breathable surface region.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in a second container containing a second breathable surface region.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second container is larger than the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the second breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the second breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the initiation of the first amplification is performed in a first container containing a first breathable surface region, and in step (c), the rapid second amplification is performed in the first container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are stacked on the first breathable surface region with an average thickness of just or about one cell layer to just or about three cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the APC is laminated onto the first breathable surface region with an average thickness of just or about two cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), APC is laminated onto the first breathable surface region with an average thickness of just or about the following: 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), the APC is laminated onto the first breathable surface region with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (c), APC is laminated onto the first breathable surface region with an average thickness of just or approximately the following: 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cell layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:9.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from a range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of exactly or about 1:2.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the preliminary first amplification is performed by supplementing the cell culture medium of the first TIL population with additional antigen-presenting cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected as exactly or greater. Approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4... 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 1.5:1 to exactly or about 100:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 50:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 25:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 20:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or approximately 10:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 50 times larger in number than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second TIL population is at least exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times larger than the first TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is supplemented with additional IL-2 exactly or about 2 days or exactly or about 3 days after the start of the second time period in step (c).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, further comprising the step of cryopreserving the collected TIL population in step (d) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, including an additional step (e) after step (d) of transferring the collected TIL population from step (d) to an infusion bag optionally containing HypoThermosol.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, comprising the step of cryopreserving an infusion bag containing the collected TIL population in step (e) using a cryopreservation process.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation process is performed using a 1:1 ratio of collected TIL populations to cryopreservation medium.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (b) is 2.5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (c) is 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a PBMC.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMC is irradiated and allogeneic.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the antigen-presenting cells are artificial antigen-presenting cells.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a membrane-based cell processing system is used for the collection in step (d).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the collection in step (d) is performed using the LOVO cell processing system.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 5 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 10 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 15 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 20 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 25 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are comprised of exactly or about 30 to exactly or about 60 segments per container in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 35 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 40 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) of exactly or about 45 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or about 50 to exactly or about 60 segments per container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments are contained in step (b) exactly or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 segments per container.

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately 27 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 20 ^mm³ to exactly or about 50 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 21 ^mm³ to exactly or about 30 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 22 ^mm³ to exactly or about 29.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 23 ^mm³ to exactly or about 29 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 24 ^mm³ to exactly or about 28.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 25 ^mm³ to exactly or about 28 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or about 26.5 ^mm³ to exactly or about 27.5 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each segment has a volume of exactly or approximately the following: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 30 to exactly or about 60 segments, wherein the total volume is exactly or about 1300 ^mm³ to exactly or about 1500 ^mm³ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total volume is exactly or about 1350 ^mm³ .

In other embodiments, the invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a plurality of segments comprises exactly or about 50 segments, wherein the total mass is exactly or about 1 gram to exactly or about 1.5 grams.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cell culture medium is provided in a container in the form of a G container or a Xuri cell bag.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is from about 10,000 IU/mL to about 5,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the IL-2 concentration in the cell culture medium is about 6,000 IU/mL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second time period in step (c) is performed within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 5 days, 6 days, or 7 days, respectively.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each carried out within a time period of exactly or approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 16 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 17 days to exactly or about 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 14 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total period of exactly or about 15 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 16 days to exactly or about 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 14 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed over a total of exactly or about 15 days to exactly or about 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 14 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 15 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) through (d) are performed in a total of exactly or approximately 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 14 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 15 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 16 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein steps (a) to (d) are performed in a total time of exactly or about 18 days or less.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the therapeutic TIL population collected in step (d) contains a therapeutically effective dose of TIL sufficient for use with TIL.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of TILs sufficient for a therapeutically effective dose is exactly or about 2.3 × 10¹⁰ to exactly or about 13.7 × 10¹⁰.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 16 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 17 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the third TIL population in step (c) provides at least one to five times or more of interferon-γ production compared to TILs prepared by a process longer than 18 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein effector T cells and/or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression relative to effector T cells and/or central memory T cells obtained from the second cell population in step (b).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a closed container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a G container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-10.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-100.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein each container referenced in the method is a GREX-500.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared by the methods described in any of the foregoing paragraphs as applicable above.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs) or OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any antigen-presenting cells (APCs).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of any OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a method of first expansion of TILs without the addition of antigen-presenting cells (APCs) and OKT3.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 16 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 17 days.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient’s tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production, and/or increased polyclonalness compared to TILs prepared by a process of length exceeding 18 days.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased interferon-γ production.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased polyclonalness.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs as applicable above, the therapeutic TIL population providing increased efficacy.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A through F above, or according to steps A through F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than one times the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least twice the amount of interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least more than twice the amount of interferon-γ.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 16 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 17 days. In other embodiments, the present invention provides a therapeutic TIL population as described in any of the foregoing paragraphs, modified to produce at least three times more interferon-γ compared to TILs prepared by a process longer than 18 days. In some implementations, the amplification process described herein, such as in steps A to F above or according to steps A to F above (also as shown, for example, in Figure 8 (especially, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)) enables TIL to produce at least three times more interferon-γ.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any antigen-presenting cells (APCs). In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are made capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that is capable of producing at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are capable of producing at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least twice as much interferon-γ as TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least twice as much interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any APC. In some embodiments, the TILs are able to produce at least one times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a therapeutic population of TILs that produces at least three times more interferon-γ compared to TILs prepared by a method of first amplification of TILs without the addition of any OKT3. In some embodiments, the TILs are able to produce at least three times more interferon-γ due to the amplification process described herein, such as in or according to steps A through F above (also as shown, for example, in Figure 8 (particularly, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G and/or Figure 8H and/or Figure 8I and/or Figure 8J and/or Figure 8K and/or Figure 8L and/or Figure 8M and/or Figure 8N and/or Figure 8O and/or Figure 8P)).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy), a core needle biopsy, a core needle puncture biopsy, or a fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a fine-needle aspirate.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the tumor fragment is obtained by core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirates; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first amplification step; (iii) the method includes a period of approximately 8 days for initiating the first amplification; and (iv) the method includes a period of approximately 11 days for rapid second amplification. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population from one or more tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating the first expansion step; (iii) the method includes a period of approximately 8 days for initiating the first expansion; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing the subcultures for approximately 6 days. In some of the foregoing embodiments, up to 5 subcultures are cultured separately in containers of the same size or larger as the container in which the second TIL population is initially cultured in the rapid second expansion. In some of the foregoing embodiments, the culture of the second TIL population is evenly distributed among up to 5 subcultures. In some of the aforementioned embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a subject, including small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a fine-needle aspirate obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1 to 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1 to about 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first TIL population is a small biopsy (including, for example, a perforated biopsy) obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a subject.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step to obtain a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days; and (iv) the method includes a rapid second expansion step by culturing the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 11 days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method includes obtaining a first TIL population by core needle biopsy of 1 to 10 tumor tissues from a subject; (ii) the method includes a step of culturing the first TIL population in a cell culture medium containing IL-2 for approximately 3 days prior to initiating a first expansion step; (iii) the method includes initiating a first expansion step by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) for approximately 8 days to obtain a second TIL population; and (iv) the method includes performing a rapid second expansion by culturing a culture of the second TIL population in a cell culture medium containing IL-2, OKT-3, and APCs for approximately 5 days, splitting the culture into up to 5 subcultures, and culturing each of the subcultures in a cell culture medium containing IL-2 for approximately 6 days. In some of the foregoing embodiments, up to five subcultures are cultured in containers of the same size or larger than the container in which the second TIL population was started during the rapid second amplification. In some of the foregoing embodiments, the cultures of the second TIL population are evenly distributed among up to five subcultures. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein (i) the method comprises obtaining a first TIL population from 1 to about 10 tumor tissue samples from a subject by core needle biopsy; (ii) the method comprises, prior to performing the first amplification step, culturing the first TIL population in a G-REX-100M culture flask in a cell culture medium containing 0.5 L CM1 medium of 6000 IU IL-2/mL for about 3 days; (iii) the method comprises initiating the first amplification by adding 0.5 L CM1 medium containing 6000 IU/mL IL-2, 30 ng/mL OKT-3, and about ^10⁸ feeder cells, and culturing for about 8 days; and (iv) the method comprises performing a rapid second amplification by transferring the second TIL population to a 5 L CM1 medium containing 3000 IU/mL IL-2, 30 ng/mL OKT-3, and 5 × ^10⁹ feeder cells. (a) The culture is divided into up to five subcultures by transferring ^10⁹ TILs into each of up to five G-REX-500MCS culture flasks containing 5 L AIM-V medium with 3000 IU/mL IL-2, and said subcultures are cultured for approximately six days. In some of the foregoing embodiments, the steps of the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (a) initiating a first expansion of a first T cell population obtained from a donor by culturing a first T cell population to achieve growth and initiate activation of the first T cell population; (b) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (c) collecting the second T cell population. In other embodiments, the rapid second expansion step is broken down into multiple steps to achieve a vertical scaling up of culture scale by: (a) rapidly expanding the first T cell population by culturing it in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve lateral scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 4 to 7 days. In other embodiments, the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second expansion step is broken down into multiple steps to achieve a longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring the first T cell population from the small-scale culture to a second container (e.g., a G-REX-500MCS container) larger than the first container, and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second container for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a first small-scale culture in a first container (e.g., a G-REX-100MCS container) for a period of approximately 2 to 4 days; and then (b) transferring and allocating the first T cell population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, wherein in each second container, a portion of the first T cell population transferred from the first small-scale culture is cultured in the second small-scale culture for a period of approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 2 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve both lateral and longitudinal scaling up of the culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture into 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 to 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 5 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and allocating the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid expansion step is broken down into multiple steps to achieve lateral and longitudinal scaling up of culture scale by: (a) performing a rapid second expansion by culturing a first T cell population in a small-scale culture in a first container (e.g., a G-REX-100MCS container) for approximately 3 to 4 days; and then (b) transferring and distributing the first T cell population from the small-scale culture to at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) that are larger than the first container, wherein in each second container, a portion of the first T cell population transferred from the small-scale culture is cultured in a larger-scale culture for approximately 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of up to 7 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed over a period of up to 11 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over a period of 7 or 8 days, and the rapid second amplification in step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the initiation of the first amplification in step (a) is performed over an 8-day period, and the rapid second amplification in step (b) is performed over a period of up to 8 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), the first T cell population is cultured in a first culture medium containing OKT-3 and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises a 4-1BB agonist, OKT-3, and IL-2.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), the first T cell population is cultured in a second culture medium containing OKT-3, IL-2 and antigen-presenting cells (APC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second culture medium comprises 4-1BB agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains 4-1BB agonist, OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous as a donor for the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2 and a second APC population, wherein the second APC population is exogenous as a donor for the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first T cell population is cultured in a first culture medium in a container containing a first breathable surface, wherein the first culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC population is stacked on the first breathable surface; wherein in step (b), the first T cell population is cultured in a second culture medium in a container, wherein the second culture medium contains a 4-1BB agonist, OKT-3, IL-2, and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is stacked on the first breathable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the ratio of the number of APCs in the second APC group to the number of APCs in the first APC group is about 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the number of APCs in the first APC group is about 2.5 × ^10⁸ and the number of APCs in the second APC group is about 5 × ^10⁸ .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is laminated onto a first breathable surface with an average thickness of two APC layers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC group is laminated onto the first breathable surface with an average thickness selected from 4 to 8 APC layers.

In other embodiments, the present invention provides a modified version of the method described in any of the foregoing paragraphs as applicable above, wherein the ratio of the average number of APC layers stacked on the first breathable surface in step (b) to the average number of APC layers stacked on the first breathable surface in step (a) is 2:1.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.0 × ^10⁶ APCs/ ^cm² to just or about 4.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 1.5 × ^10⁶ APCs/ ^cm² to just or about 3.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density selected from just or about 2.0 × ^10⁶ APCs/ ^cm² to just or about 3.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC population is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 2.5 × ^10⁶ APCs/ ^cm² to just or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from just or about 3.5 × ^10⁶ APCs/ ^cm² to just or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (b), a second APC population is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.0 × ^10⁶ APCs/ ^cm² to exactly or about 4.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 2.5 × ^10⁶ APCs/ ^cm² to exactly or about 7.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 1.5 × ^10⁶ APCs/ ^cm² to exactly or about 3.5 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 3.5 × ^10⁶ APCs/ ^cm² to exactly or about 6.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density selected from exactly or about 2.0 × ^10⁶ APCs/ ^cm² to exactly or about 3.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density selected from exactly or about 4.0 × ^10⁶ APCs/ ^cm² to exactly or about 5.5 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein in step (a), a first APC group is seeded on a first breathable surface at a density of exactly or about 2.0 × ^10⁶ APCs/ ^cm² , and in step (b), a second APC group is seeded on the first breathable surface at a density of exactly or about 4.0 × ^10⁶ APCs/ ^cm² .

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the APC is a peripheral blood mononuclear cell (PBMC).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the PBMCs are irradiated and the donor for the first T cell population is exogenous.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are bone marrow infiltrating lymphocytes (MIL).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cells are peripheral blood lymphocytes (PBLs).

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by separation from whole blood of a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained by isolating from the donor's apheresis product.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a first T cell population is isolated from whole blood or apheresis products of a donor by positive or negative selection of T cell phenotype.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the T cell phenotype is CD3+ and CD45+.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs, wherein T cells are isolated from NK cells prior to initiating a first expansion of a first T cell population. In other embodiments, T cells in a first T cell population are separated from NK cells by removing CD3-CD56+ cells from the first T cell population. In other embodiments, CD3-CD56+ cells are removed from the first T cell population by using a gating strategy that removes CD3-CD56+ cell fractions and recovers negative fractions. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of NK cells. In other embodiments, the foregoing method is used for T cell expansion in a first T cell population characterized by a high percentage of CD3-CD56+ cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of NK cells. In other embodiments, the foregoing method is used for T cell expansion in tumor tissue characterized by a large number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of NK cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with a tumor characterized by a high number of CD3-CD56+ cells. In other embodiments, the aforementioned method is used for T cell expansion from tumor tissue obtained from a patient with ovarian cancer.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein exactly or about 1 × ^10⁷ T cells from a first T cell population are seeded into a container to initiate an initial first expansion culture in such a container.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs applicable above, wherein a first T cell population is distributed into a plurality of containers, and exactly or about 1 × 10⁷ T cells from the first T cell population are seeded in each container to initiate an initial first expansion culture in such containers.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the second T cell population collected in step (c) is a therapeutic TIL population.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via small biopsy (including, for example, perforated biopsy), core needle biopsy, core needle puncture biopsy, or fine needle aspiration.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies, or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue samples from a donor, including small biopsies (such as perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies), core needle biopsies, core needle puncture biopsies or fine needle aspirations from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, perforated biopsies) of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue samples obtained from a donor via core needle biopsy.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsy of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissues from a donor.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the first T cell population is obtained from core needle biopsies of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tumor tissues from a donor.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsies, core needle biopsies, or needle biopsies obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the initiation of the first expansion is performed in a container containing a first breathable surface region, wherein the initiation of the first expansion is performed for a first time period of approximately 7 or 8 days to obtain the second TIL population. (iii) A second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iv) a rapid second expansion is performed by supplementing the second TIL population with a second cell culture medium containing additional IL-2, OKT-3 and APC to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (ii), wherein the rapid second expansion is performed for a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container containing a second ventilated surface area; (v) the therapeutic TIL population obtained from step (iii) is collected; and (v) the collected TIL population from step (iv) is transferred to an infusion bag.

In other embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining and/or receiving a first TIL population from one or more small biopsy, core needle biopsy, or needle biopsy samples obtained from a tumor in a subject by culturing a tumor sample in a first cell culture medium containing IL-2 for approximately 3 days; and (ii) initiating a first expansion by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a second TIL population, wherein the initiation... The first amplification is carried out over a first period of approximately 7 or 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (iii) a rapid second amplification is carried out by contacting the second TIL population with a third cell culture medium containing IL-2, OKT-3 and APC to produce a third TIL population, wherein the rapid second amplification is carried out over a second period of approximately 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second amplification is carried out in a container containing a second ventilated surface area; and (iv) the therapeutic TIL population obtained from step (iii) is collected.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with an additional amount of third culture medium and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein after day 5 of the second time period, the culture is split into two or more subcultures, and each subculture is supplemented with a fourth medium containing IL-2 and cultured for approximately 6 days.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the culture is split into up to five subcultures after day 5 of the second time period.

In other embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein all steps in the method are completed within approximately 22 days.

In other embodiments, the present invention provides a method for expanding T cells, the method comprising: (i) initiating a first expansion of a first T cell population by culturing a first T cell population to achieve growth and initiate activation of the first T cell population, the first T cell population being derived from a tumor sample obtained from one or more small biopsies, core needle biopsies, or needle biopsies of a tumor in a donor; (ii) after the activation of the first T cell population initiated in step (a) begins to decay, rapidly expanding the first T cell population a second T cell population by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; and (iv) collecting the second T cell population. In some embodiments, the tumor sample is obtained from multiple core needle biopsies. In some embodiments, the multiple core needle biopsies are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, and 10 core needle biopsies.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein T cells or TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubating the tumor in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, California). In some embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes one or more dissociation (digestive) enzymes, such as (but not limited to) collagenase (including any blended or type of collagenase), Accutase ^™ , Accumax ^™ , hyaluronidase, neutral protease (dispersant enzyme), chymotrypsin, papain, trypsin, casein, elastase, papain, protease type XIV (streptomycin), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation or proteolytic enzymes, and any combination thereof. In other embodiments, the tumor is placed in a mixture of tumor dissociation enzymes, which includes collagenase (including any blended or type of collagenase), neutral protease (dispersant enzyme), and deoxyribonuclease I (DNase).

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a TIL population undergoes a gene-editing process. In some embodiments, the gene-editing process may be performed at any time during the TIL amplification process, including before, during, or after any step of the amplification process, such as before, during, or after one or more of steps A, B, C, D, E, and F outlined above.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein an activation step follows a first amplification step. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and/or anti-CD38 beads for about 1-7 days. In some embodiments, the activation step includes culturing TILs in a medium containing anti-CD3 and anti-CD38 beads for about 1-7 days. In some embodiments, the activation step is performed for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the activation step is followed by a gene editing step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a gene editing step follows a first amplification step. In some embodiments, when the first amplification step includes OKT-3, a gene editing step follows the first amplification step.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein the gene editing step includes a sterile electroporation step on a TIL population. In some embodiments, the sterile electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein. According to some embodiments, the TALE nuclease system downregulates the expression of PD-1. According to some embodiments, the gene editor further comprises a TALE nuclease system that downregulates the expression of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some embodiments, the resulting TIL is a PD-1 gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CTLA-4 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/LAG-3 double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/CISH double gene knockout TIL. According to some embodiments, the resulting TIL is a PD-1/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a PD-1/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/LAG-3 double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CTLA-4/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CISH double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a LAG-3/CBL-B double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/TIGIT double gene knockout TIL. According to some implementation schemes, the obtained TIL is a CISH/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL is a TIGIT/CBL-B double gene knockout TIL. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CTLA-4. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and TIGIT. According to some implementation schemes, the resulting TIL exhibits downregulated expression of PD-1 and CBL-B. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and LAG-3. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and CISH. According to some implementation schemes, the resulting TIL exhibits downregulated expression of CTLA-4 and TIGIT. According to some implementation schemes, the obtained TILs showed downregulation of CTLA-4 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CISH expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of LAG-3 and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and TIGIT expression. According to some implementation schemes, the obtained TILs showed downregulation of CISH and CBL-B expression. According to some implementation schemes, the obtained TILs showed downregulation of TIGIT and CBL-B expression.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a quiescent step follows the gene editing step. In some embodiments, the quiescent step comprises incubating a TIL population at about 30-40°C and about 5% _CO2 for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 30°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1-24 hours. According to some embodiments, the quiescent step is performed at about 37°C for about 1 hour, followed by about 15-23 hours at about 30°C.

In some embodiments, the present invention provides a modified method as described in any of the foregoing paragraphs as applicable above, wherein a second amplification step follows the resting step.

VIII. Pharmaceutical Compositions, Dosage, and Dosing Regimens

In some embodiments, TILs, MILs, or PBLs (including TILs, MILs, or PBLs genetically modified to express CCR) amplified and/or genetically modified using the methods of this disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC or melanoma. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is metastatic NSCLC or metastatic melanoma. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic melanoma. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of NSCLC. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly in the case of metastatic NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

In other embodiments, the present invention provides an infusion bag that includes the therapeutic TIL population described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a pharmaceutically acceptable carrier as described in any of the preceding paragraphs.

In other embodiments, the present invention provides an infusion bag comprising the TIL composition described in any of the preceding paragraphs.

In other embodiments, the present invention provides a cryopreservation formulation for a therapeutic TIL population as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population and a cryopreservation culture medium as described in any of the preceding paragraphs.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains DMSO.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a cryopreservation formulation of the TIL composition described in any of the preceding paragraphs.

In some embodiments, TILs amplified using the methods of this disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, the T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs are administered, with an average of about 7.8 × ^10¹⁰ TILs, particularly in cases where the cancer is NSCLC. In some embodiments, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs, particularly when the cancer is NSCLC. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9×10⁶, 1× ^10⁷ , 2× ^10⁷ , 3× ^10⁷ , 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7× ^10⁷ , 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , 1× ^10⁹ , 2 ^{× 10⁹} 3× ^10⁹ , 4× ^10⁹ , 5×10⁹, 6× ^10⁹ , 7× ^10⁹ , 8× ^10⁹ , 9× ^10⁹ , 1× ^10¹⁰ , 2× ^10¹⁰ , 3× ^10¹⁰ , 4× ^10¹⁰ , 5× ^10¹⁰ , 6× ^10¹⁰ , 7× ^10¹⁰ , 8× ^10¹⁰ , 9× ^10¹⁰ , 1× ^10¹¹ , 2× ^10¹¹ , 3× ^10¹¹ , 4× ^10¹¹ , 5× ^10¹¹ , 6× ^10¹¹ , 7× ^10¹¹ , 8× ^10¹¹ , 9× ^10¹¹ , 1× ^10¹² , 2× ^10¹² , 3 ^{× 10¹²} 4× ^10¹² , 5× ^10¹² , 6×10¹², 7× ^10¹² , 8× ^10¹² , 9× ^10¹² , 1× ^10¹³ , 2× ^10¹³ , 3× ^10¹³ , 4× ^10¹³ , 5× ^10¹³ , 6× ^10¹³ , 7× ^10¹³ , 8× ^10¹³ and 9 ^{× 10¹³} . In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% of the pharmaceutical composition. %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50% of the pharmaceutical composition. 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. The range of about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL in the pharmaceutical composition provided by the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.2g. 5g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, or 0.0025g. , 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007 g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.4 5g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form of compound administration, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. A clinically determined dosage of the TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the condition or disease, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some implementations, TIL can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some implementations, TIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. TIL administration can be continued as needed.

In some implementation schemes, the effective dose of TIL is approximately 1× ^10⁶ , 2× ^10⁶ , ³ ×10⁶, 4× ^10⁶ , 5× ^10⁶ , 6×10⁶, 7× ^10⁶ , ⁸ × ^{10⁶, 9×10⁶ ,} 1× ^10⁷ , 2×10⁷, 3× ^10⁷ , ⁴ × ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , ⁹ × ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3×10⁸, 4× ^10⁸ , ⁵ × ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8×10⁸, 9× ^10⁸ , ¹ × ^10⁹ , 2× ^10⁹ , 3×10⁹. ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some implementations, the effective dose of TIL is in the range of 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose of TIL is from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, and from about 0.7 mg/kg to about The range is from 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, and so on. The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

Effective amounts of TILs can be administered via any recognized mode of administration of a reagent with similar efficacy, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation, in single or multiple doses.

IX. Methods of treating patients

The treatment method begins with the collection and culture of raw TILs. Such methods have been described in the art, for example, by reference in their entirety to Jin et al., J. Immunotherapy, 2012, 35(3):283-292. The implementation of the treatment method is described throughout the following sections, including examples.

The methods described herein, including, for example, those described in steps A through F above or those described in steps A through F above (and also, for example, those shown in Figures 1, 36, and/or 8), have been found to have specific uses in the treatment of cancer patients (e.g., as described in Goff et al., J. Clinical Oncology, 2016, 34(20):2389-239 and the Supplement, which are incorporated herein by reference in their entirety). In some embodiments, TILs are grown from resected metastatic melanoma remnants as previously described (see Dudley et al., J Immunother., 2003, 26:332-342, which are incorporated herein by reference in their entirety). Fresh tumors can be segmented under sterile conditions. Representative samples can be collected for formal pathological analysis. Single fragments of 2 ^mm³ to ³ mm³ can be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained from each patient. In some embodiments, 24 samples are obtained from each patient. Samples can be placed in individual wells of a 24-well plate, maintained in growth medium containing a high dose of IL-2 (6,000 IU/mL), and tumor destruction and/or TIL proliferation can be monitored. As described herein, any tumor cells remaining viable after treatment can be enzymatically digested into a single-cell suspension and cryopreserved.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumors when available. TILs are considered reactive if overnight co-culture produces interferon-γ (IFN-γ) levels >200 pg/mL and twice the background level. (Goff et al., J Immunother., 2010, 33:840-847; incorporated herein by reference in its entirety). In some embodiments, cultures demonstrating autoreactivity or adequate growth patterns may be selected for secondary amplification (e.g., the secondary amplification provided according to step D of Figures 1, 36, and/or 8), including what is sometimes referred to as rapid amplification (REP). In some embodiments, amplified TILs exhibiting high autoreactivity (e.g., high proliferation during secondary amplification) are selected for additional secondary amplification. In some implementations, TILs with high self-reactivity (e.g., high proliferation during the second amplification provided in step D of Figures 1, 36 and/or 8) are selected for additional second amplification according to step D of Figures 1, 36 and/or 8.

Cell phenotypes of cryopreserved samples of infusion bag TILs can be analyzed by flow cytometry targeting surface markers CD3, CD4, CD8, CCR7, and CD45RA (e.g., FlowJo) (BD BioSciences) and by any of the methods described herein. Serum cytokines were measured using standard enzyme-linked immunosorbent assay (ELISA). An increase in serum IFN-γ was defined as a baseline level >100 pg/mL and greater than 43.

In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, achieve remarkable improvements in clinical efficacy. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1, 36, and/or 8, exhibit improved clinical efficacy compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8). In some embodiments, methods other than those described herein include methods referred to as Process 1C and/or Gen 1. In some embodiments, the increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by the methods provided herein, such as those exemplified in Figures 1 or 36, exhibit similar response time and safety profiles compared to TILs produced by methods other than those described herein (including methods other than those exemplified in Figures 1, 36, and/or 8).

In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of a subject treated with TIL indicates active TIL. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TIL of a subject treated with TIL prepared by the methods of the present invention, including, for example, those described in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TIL produced by the methods of the present invention. In some embodiments, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or compared to patients treated with TIL prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is tripled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is quadrupled compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ secretion is increased fivefold compared to untreated patients and/or compared to patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared using the methods of the present invention, including those described, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ is measured in the serum of subjects treated with TILs prepared using the methods of the present invention, including those prepared, for example, in Figures 1, 36, and/or 8. In some embodiments, IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy in treating tumors.

In some embodiments, the TILs prepared by the methods of the present invention include those described, for example, in Figure 1 or Figure 36, where IFN-γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of subjects treated with TILs indicates active TILs. In some embodiments, an efficacy assay for IFN-γ production is employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or ex vivo TILs of subjects treated with TILs prepared by the methods of the present invention, including methods described, for example, in Figures 1, 36, and/or 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TILs produced by the methods of the present invention. In some implementations, IFN-γ is increased by one, two, three, four, or five times or more compared to untreated patients and/or patients treated with TILs prepared using methods different from those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, and/or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, and/or 8, such as those referred to as the process 1C method). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, those other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, and/or 8).

Measures of efficacy may include disease control rate (DCR) and overall response rate (ORR), as known in the art and described herein.

A. Methods of treating cancer

The compositions and methods described herein can be used in a method of treating a disease. In some embodiments, they are used to treat hyperproliferative conditions, such as cancer, in adult or pediatric patients. They can also be used to treat other conditions as described herein and in the following paragraphs.

In some implementations, hyperplasia is defined as cancer. In some implementations, hyperplasia is defined as solid tumor cancer. In some implementations, solid tumor cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papillomavirus (HPV), central nervous system-related cancers (including ependymoma, neuroblastoma, neuroblastoma, pineal blastoma, and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSC)). C) Hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, and pharyngeal cancer; kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC), metastatic NSCLC, and small cell lung cancer); melanoma (including uveal melanoma, choroidal melanoma, ciliary body melanoma, iris melanoma, or metastatic melanoma); mesothelioma (including malignant pleural mesothelioma); ovarian cancer; pancreatic cancer (including pancreatic duct adenocarcinoma); penile cancer; rectal cancer; kidney cancer; renal cell carcinoma; sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas); thyroid cancer (including anaplastic thyroid cancer); uterine cancer and vaginal cancer.

In some embodiments, the hyperplastic condition is a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from the group consisting of: chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-B, wherein the cancer is a hematologic malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a modified MIL or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-BR, wherein the cancer is a hematologic malignancy.

In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, that is relapsed or refractory to treatment with at least one prior therapy (including chemotherapy, radiotherapy, or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least two prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy). In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least three prior therapies (including chemotherapy, radiotherapy, and/or immunotherapy).

In some embodiments, the cancer is a microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. MSI-H and dMMR cancers and their detection have been described in Kawakami et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosure of which is incorporated herein by reference.

In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a non-human. In some embodiments, the present invention includes a method of treating a patient with cancer using a modified TIL, MIL, or PBL that downregulates one or more of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, wherein the patient is a companion animal.

In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors and/or MEK inhibitors. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method for treating a patient with cancer that is difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method of treating a patient with cancer that is difficult to treat with BRAF inhibitors selected from the group consisting of vemurafenib, dabrafenib, enrafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof; and difficult to treat with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, bemetinib, selmetinib, pimatetinib, refatinib, and pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is a pediatric cancer.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the cancer is uveal melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the uveal melanoma is a choroidal melanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is neuroblastoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the pediatric cancer is a central nervous system (CNS) related cancer. In some embodiments, the pediatric cancer is difficult to treat with chemotherapy. In some embodiments, the pediatric cancer is difficult to treat with radiation therapy. In some embodiments, the pediatric cancer is difficult to treat with dinutuximab.

In some embodiments, the present invention includes a method for treating a patient with cancer, wherein the CNS-related cancer is neuroblastoma, pinealoblastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein can be used to treat cancer that is refractory to anti-PD-1 or anti-PD-L1 antibodies or resistant to prior treatment. In some embodiments, the patient is a primary refractory patient to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has not shown a prior response to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has shown a prior response to anti-PD-1 or anti-PD-L1 antibodies, followed by cancer progression. In some embodiments, the cancer is refractory to combination therapy with anti-CTLA-4 antibodies and/or anti-PD-1 or anti-PD-L1 antibodies in combination with at least one chemotherapeutic agent. In some embodiments, the prior chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and/or cisplatin. In some prior embodiments, the chemotherapeutic agent is a platinum-based dual chemotherapeutic agent. In some embodiments, the platinum dual therapy comprises a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel, docetaxel, or nab-paclitaxel). In some embodiments, the platinum dual chemotherapeutic agent is combined with pemetrexed.

In some implementations, NSCLC is PD-L1 negative and/or comes from patients with cancers that express PD-L1 and have a tumor proportion score (TPS) <1%, as described elsewhere in this article.

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some implementations, NSCLC is difficult to treat with combination therapy comprising an anti-PD-1 antibody or anti-PD-L1 antibody, pemetrexed, and platinum dual therapy, wherein the platinum dual therapy comprises:

i) The first-line chemotherapy agent, selected from the group consisting of cisplatin and carboplatin.

ii) and a second chemotherapy agent selected from the group consisting of: vinorelbine, gemcitabine and taxanes (including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel).

In some embodiments, NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient has not received treatment. In some embodiments, NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, NSCLC has been previously treated with a chemotherapy agent. In some embodiments, NSCLC has been previously treated with a chemotherapy agent but is no longer being treated with said chemotherapy agent. In some embodiments, the NSCLC patient has not received anti-PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the NSCLC patient has not received NSCLC treatment or has received chemotherapy agent treatment but has not received PD-1/PD-L1 treatment. In some embodiments, the NSCLC patient has not received treatment or has received chemotherapy agent treatment but has not received anti-PD-1/PD-L1 treatment and has low PD-L1 expression. In some embodiments, the NSCLC patient had massive disease at baseline. In some embodiments, the subject has massive disease and low PD-L1 expression at baseline. In some embodiments, the NSCLC patient does not have detectable PD-L1 expression. In some embodiments, the NSCLC patient is untreated or has received chemotherapy but not anti-PD-1/PD-L1 treatment and does not have detectable PD-L1 expression. In some embodiments, the patient has massive disease and does not have detectable PD-L1 expression at baseline. In some embodiments, the NSCLC patient has untreated NSCLC or has received chemotherapy (e.g., received chemotherapy agents) but not anti-PD-1/PD-L1 treatment, and the patient has low PD-L1 expression and/or has massive disease at baseline. In some embodiments, massive disease is indicated when the maximum tumor diameter measured in the transverse or coronal plane is greater than 7 cm. In some embodiments, massive disease is indicated when there are swollen lymph nodes with a short axis diameter of 20 mm or greater. In some embodiments, the chemotherapy agents include standard of care treatments for NSCLC.

In some embodiments, PD-L1 expression is determined by a tumor proportion score. In some embodiments, a subject with refractory NSCLC has a tumor proportion score (TPS) of <1%. In some embodiments, a subject with refractory NSCLC has a TPS of ≥1%. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject with refractory NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment.

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in Figures 1, 36, or 8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not exemplified in Figures 1, 36, or 8, including, for example, methods referred to as Process 1C). In some embodiments, significantly enhanced polyclonalness and/or increased polyclonalness indicate therapeutic efficacy and/or increased clinical efficacy in cancer treatment. In some embodiments, polyclonalness refers to T-cell reservoir diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to the administration of TILs prepared by the methods of the present invention. In some embodiments, polyclonalness is increased by one, two, ten, 100, 500, or 1000 times compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is doubled compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is tenfold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonality is 100fold increased compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8). In some embodiments, polyclonalness is increased by 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in Figures 1, 36, or 8).

In some embodiments, PD-L1 expression is determined by tumor proportion scoring using one or more assays as described herein. In some embodiments, the subject or patient with NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the NSCLC tumors have a TPS of ≥1%. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and the tumor proportion score was determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with NSCLC has previously been treated with anti-PD-L1 antibodies and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC tumors has a TPS of ≥1%. In some embodiments, the subject or patient with refractory or drug-resistant NSCLC has previously been treated with anti-PD-1 and/or anti-PD-L1 antibodies and a tumor proportion score has been determined prior to anti-PD-1 and/or anti-PD-L1 antibody treatment.

In some implementations, NSCLC is NSCLC exhibiting a tumor proportion score (TPS), or the percentage of live tumor cells obtained from the patient prior to anti-PD-1 or anti-PD-L1 therapy, or the percentage of PD-L1 protein showing partial or complete membrane staining at any intensity of less than 1% (TPS<1%). In some implementations, NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, <0.04%, <0.03%, <0.02%, and <0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS selected from the group consisting of: about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, and about 0.01%. In some embodiments, an NSCLC is an NSCLC exhibiting a TPS between 0% and 1%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.9%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.8%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.7%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.6%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.5%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.4%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.3%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.2%. In some embodiments, the NSCLC exhibits a TPS between 0% and 0.1%. TPS can be measured by methods known in the art, such as those described in Hirsch et al., J. Thorac. Oncol. 2017, 12, 208-222, or methods used to determine TPS prior to treatment with pembrolizumab or other anti-PD-1 or anti-PD-L1 therapies. Methods approved by the U.S. Food and Drug Administration for measuring TPS can also be used. In some embodiments, PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

In some embodiments, partial membrane staining includes 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more. In some embodiments, complete membrane staining includes approximately 100% membrane staining.

In some implementations, testing PD-L1 may involve measuring the level of PD-L1 in a patient's serum. In these implementations, measuring PD-L1 in a patient's serum eliminates the uncertainty of tumor heterogeneity and the discomfort of patients undergoing serial biopsies.

In some implementations, elevated soluble PD-L1 levels compared to baseline or standard levels are associated with a prognostic risk of NSCLC progression. See, for example, Okuma et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli et al., Oncotarget, 2018, 9, 17554-17563. In some implementations, PD-L1 is exosomal PD-L1. In some implementations, PD-L1 is expressed on circulating tumor cells.

In some implementation schemes, the subject or patient has non-small cell lung cancer (NSCLC), characterized by at least one of the following:

i. PD-L1 pre-defined tumor proportion score (TPS) <1%,

ii. The TPS score of PD-L1 is 1%-49%, or

iii. The predetermined absence of one or more driver mutations,

The driving mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20 mutations, KRAS mutations, BRAF mutations, ALK mutations, c-ROS mutations (ROS1 mutations), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and/or altered MET signals, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations, and GNA11 mutations.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population described herein.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition described herein.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to the administration of therapeutically effective doses of the therapeutic TIL population and TIL composition described herein, respectively.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, the method further comprising the step of initiating treatment of the subject with a high-dose IL-2 regimen the day after administration of TIL cells to the subject.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg every eight hours via a 15-minute bolus intravenous infusion until tolerated.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from the cancer described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is glioblastoma (including GBM).

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified method for treating a subject suffering from a cancer described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides a therapeutic TIL population as described herein in a method for treating a subject suffering from cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides a method for treating a subject suffering from cancer using a TIL composition described herein, comprising administering a therapeutically effective dose of the TIL composition to the subject.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administration of a therapeutically effective dose of the therapeutic TIL population or TIL composition described herein.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, which further includes the step of starting treatment of the patient with a high-dose IL-2 regimen the day after administration of TIL cells.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein a high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg via intravenous infusion every eight hours in the form of 15-minute bolus injections until tolerated.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a solid tumor.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is metastatic melanoma.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is metastatic NSCLC.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is glioblastoma.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is a highly mutated cancer.

In other embodiments, the present invention provides a modified therapeutic population or composition of TILs described herein, wherein the cancer is a pediatric high-mutation cancer.

In other embodiments, the present invention provides the use of the therapeutic TIL population described herein in a method of treating a subject’s cancer, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

In other embodiments, the present invention provides the use of the TIL composition described in any of the foregoing paragraphs in a method of treating a subject’s cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In other embodiments, the present invention provides the use of the therapeutic TIL population or the TIL composition described herein in a method of treating a patient with cancer, the method comprising administering a non-myeloablative lymphocyte depletion regimen to the patient and then administering to the subject a therapeutically effective dose of the therapeutic TIL population or the therapeutically effective dose of the TIL composition described herein in any of the preceding paragraphs.

1. Combination with PD-1 and PD-L1 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, which includes TIL and one or more PD-1 and/or PD-L1 inhibitors.

Programmed death 1 (PD-1) is a 288-amino acid transmembrane immune checkpoint receptor protein expressed by T cells, B cells, natural killer (NK) T cells, activated monocytes, and dendritic cells. PD-1, also known as CD279, belongs to the CD28 family and is encoded by the Pdcd1 gene on chromosome 2 in humans. PD-1 consists of an immunoglobulin (Ig) superfamily domain, a transmembrane domain, and an intracellular domain containing the immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoreceptor tyrosine switching motif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play important roles in immune tolerance, as described by Keir et al., Annu. Rev. Immunol. 2008, 26, 677-704. PD-1 provides a suppressive signal that negatively regulates T cell immune responses. PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273) are expressed on tumor cells and stromal cells, which may encounter activated T cells expressing PD-1, leading to immunosuppression of T cells. PD-L1 is a 290-amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9. Blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2 using PD-1 inhibitors, PD-L1 inhibitors, and/or PD-L2 inhibitors can overcome immune resistance, as demonstrated in recent clinical studies, such as those described in Topalian et al., N. Eng. J. Med. 2012, 366, 2443-54. PD-L1 is expressed on many tumor cell lines, while PD-L2 is primarily expressed on dendritic cells and some tumor lines. In addition to T cells (which induce PD-1 expression upon activation), PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes, and dendritic cells.

In some embodiments, the PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail below. Regarding PD-1 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, references herein to PD-1 inhibitors as antibodies may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, references herein to PD-1 inhibitors may also refer to small molecule compounds or their pharmaceutically acceptable salts, esters, solvates, hydrates, cocrystals, or prodrugs.

In some embodiments, the PD-1 inhibitor is an antibody (i.e., an anti-PD-1 antibody) or a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In some embodiments, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competitively binds to PD-1 and/or binds to an epitope on PD-1. In some embodiments, the antibody competitively binds to PD-1 and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a KD of about 100 pM or less, at a KD of about 90 pM or less, at a KD of about 80 pM or less, at a KD of about 70 pM or less, at a KD of about 60 pM or less, at a KD of about 50 pM or less, at a KD of about 40 pM or less, at a KD of about 30 pM or less, at a KD of about 20 pM or less, at a KD of about 10 pM or less, or at a KD of about 1 pM or less.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 at a k _{-association rate} of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about 7.5 × ^10⁵ l/M·s or faster, at a k- _association rate of about ⁸ × 10⁵ l/M·s or faster, at a k _{-association rate} of about 8.5 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9 × ^10⁵ l/M·s or faster, at a k- _{association rate} of about 9.5 × ^10⁵ l/M·s or faster, or at a k- _{association rate} of about 1 × ^10⁶ l/M·s or faster.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, or with a k _{-dissociation rate} of about 2.1 × ^10⁻⁵ 1/s or slower, or with a k-dissociation rate of about 2.2 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.3 × 10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.6 × ^10⁻⁵ 1/s or slower, or with a k- _{dissociation rate} of about 2.7 × ^10⁻⁵ 1/s or slower. It binds to human PD-1 at a k- _{dissociation rate} of 1/s or slower, at a k- _{dissociation rate} of approximately 2.8 × ^10⁻⁵ 1/s or slower, at a k- _{dissociation rate} of approximately 2.9 × ^10⁻⁵ 1/s or slower, or at a k- _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, or about 6 nM or less. The binding of human PD-L2 to human PD-1; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower; blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower; or blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-1 inhibitor is nivolumab (available from Bristol-Myers Squibb via OPDIVO) or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-1 receptor. In some embodiments, the anti-PD-1 antibody is an immunoglobulin G4κ anti-(human CD274) antibody. Nivolumab is designated Chemical Abstracts Service (CAS) Registry No. 946414-94-4 and is also known as 5C4, BMS-936558, MDX-1106, and ONO-4538. The preparation and characteristics of nivolumab are described in U.S. Patent No. 8,008,449 and International Patent Publication No. WO 2006/121168, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of nivolumab in various forms of cancer have been described in Wang et al., Cancer Immunol. Res. 2014, 2, 846-56; Page et al., Ann. Rev. Med., 2014, 65, 185-202; and Weber et al., J. Clin. Oncology, 2013, 31, 4311-4318, the contents of which are incorporated herein by reference. The amino acid sequence of nivolumab is presented in Table 18. Nivolumab has intra-heavy chain disulfide bonds at 22-96, 140-196, 254-314, 360-418, 22”-96”, 140”-196”, 254”-314”, and 360”-418”; intra-heavy chain disulfide bonds at 23”-88”, 134”-194”’, 23”’-88”’, and 134”’-194”’; inter-heavy chain disulfide bonds at 127-214”’ and 127”-214”’; inter-heavy chain disulfide bonds at 219-219” and 222-222”; and N-glycosylation sites (H CH2 84.4) at 290” and 290”.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:158 and the light chain provided in SEQ ID NO:159. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO:159, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of nivolumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:160, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:161 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:165, SEQ ID NO:166, and SEQ ID NO:167, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of nivolumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab.

Table 18. Amino acid sequences of PD-1 inhibitors associated with nivolumab.

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at the following doses: about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 240 mg every 2 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at a dose of approximately 480 mg every 4 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma, with approximately 1 mg/kg of nivolumab administered every 3 weeks on the same day, followed by 3 mg/kg of ipilimumab for 4 consecutive doses, then 240 mg every 2 weeks or 480 mg of nivolumab every 4 weeks.

In some implementations, nivolumab is administered as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks as adjuvant therapy for melanoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks and ipilimumab at approximately 1 mg/kg every 6 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks, plus ipilimumab at 1 mg/kg every 6 weeks and two cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks or 480 mg every 4 weeks to treat metastatic non-small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat small cell lung cancer. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat small cell lung cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 360 mg every 3 weeks and ipilimumab at 1 mg/kg every 6 weeks to treat malignant pleural mesothelioma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab every 2 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at approximately 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks and 480 mg of nivolumab every 4 weeks to treat advanced renal cell carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat typical Hodgkin lymphoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat locally advanced or metastatic urothelial carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered at approximately 3 mg/kg every 2 weeks to treat pediatric patients <40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 240 mg nivolumab every 2 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab is administered at approximately 3 mg/kg, followed by ipilimumab at 1 mg/kg on the same day every 3 weeks for a total of 4 doses, then 480 mg nivolumab every 4 weeks to treat adult and pediatric patients ≥40 kg with microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, nivolumab is administered to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 240 mg every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 480 mg every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 240 mg of nivolumab is administered every 2 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab is administered at approximately 1 mg/kg, followed by 3 mg/kg ipilimumab administered on the same day every 3 weeks for 4 doses, then 480 mg of nivolumab is administered every 4 weeks to treat hepatocellular carcinoma. In some implementations, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, nivolumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 240 mg every 2 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at approximately 480 mg every 4 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor comprises pembrolizumab (available from Merck Inc. of Kenilworth, NJ, USA under the KEYTRUDA license) or its antigen-binding fragment, conjugate, or variant. Pembrolizumab is designated CAS Registry No. 1374853-91-4 and is also known as lanizumab, MK-3475, and SCH-900475. Pembrolizumab is an immunoglobulin G4 anti-(human protein PDCD1 (programmed cell death 1)) antibody containing a (human-mouse monoclonal heavy chain) disulfide bond and a human-mouse monoclonal light chain dimer structure. The structure of pembrolizumab can also be described as an immunoglobulin G4 anti-(human programmed cell death 1) antibody; containing a humanized mouse monoclonal [228-L-proline (H10-S>P)]γ4 heavy chain (134-218') disulfide bond and a humanized mouse monoclonal κ light chain dimer (226-226”:229-229”) disulfide bond. The properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO 2008/156712 A1, US Patent No. 8,354,509, and US Patent Application Publications Nos. US2010/0266617 A1, US2013/0108651A1, and US2013/0109843 A2, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of pembrolizumab in various forms of cancer are described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert et al., Lancet, 2014, 384, 1109-17; and Thomas et al., Exp. Opin. Biol. Ther., 2014, 14, 1061-1064. The amino acid sequence of pembrolizumab is illustrated in Table 19. Pembrolizumab includes the following disulfide bridges: 22-96, 22”-96”, 23”-92”’, 23”’-92”’, 134-218”, 134”-218”’, 138”-198”, 138”’-198”’, 147-203”, 147”-203”, 226-226”, 229-229”, 261-321”, 261”-321”, 367-425”, and 367”-425”; and the following glycosylation sites. (N): Asn-297 and Asn-297". Pembrolizumab is an IgG4/κ isoform containing a stable S228P mutation in the Fc region; the insertion of this mutation in the IgG4 hinge region prevents the formation of the half-molecule typically observed in IgG4 antibodies. Pembrolizumab is heteroglycosylated at Asn297 in the Fc domain of each heavy chain, resulting in a molecular weight of approximately 149 kDa for the complete antibody. The major glycoform of pembrolizumab is the fucosylated degalactosyl biantennary (GOF) form.

In some embodiments, the PD-1 inhibitor comprises the heavy chain provided in SEQ ID NO:168 and the light chain provided in SEQ ID NO:169. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy and light chain CDRs or variable regions (VRs) of pembrolizumab. In some embodiments, the PD-1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:170, and the PD-1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:171 or a conserved amino acid substitution thereof. In some embodiments, the PD-1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises _{VH and VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises VH and VL regions} , each at least 97% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:175, SEQ ID NO:176, and SEQ ID NO:177, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-1 inhibitor is a biosimilar monoclonal antibody of PD-1 approved by a regulatory agency, referencing pembrolizumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. The anti-PD-1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab.

Table 19. Amino acid sequences of PD-1 inhibitors associated with pembrolizumab.

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and nivolumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat melanoma. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat melanoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat melanoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat NSCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat NSCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat small cell lung cancer (SCLC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat SCLC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat SCLC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered to treat head and neck squamous cell carcinoma (HNSCC). In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat HNSCC. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HNSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, children are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat classic Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat urothelial carcinoma. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) cancer. In some implementations, adults are administered pembrolizumab at approximately 400 mg every 6 weeks to treat MSI-H or dMMR cancer. In some implementations, pediatrics are administered pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat MSI-H or dMMR cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient colorectal cancer (dMMR CRC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat MSI-H or dMMR CRC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat gastric cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat gastric cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat esophageal cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat esophageal cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cervical cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cervical cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat hepatocellular carcinoma (HCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat HCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are treated with pembrolizumab at approximately 200 mg every 3 weeks for Merkel cell carcinoma (MCC). In some implementations, adults are treated with pembrolizumab at approximately 400 mg every 6 weeks for MCC. In some implementations, children are treated with pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks for MCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat renal cell carcinoma (RCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks and axitinib 5 mg is administered orally twice daily to treat RCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat endometrial cancer. In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks, and lenvatinib 20 mg is administered orally once daily for non-MSI-H or dMMR tumors to treat endometrial cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, adults are given pembrolizumab at approximately 200 mg every 3 weeks to treat TMB-H cancer. In some implementations, adults are given pembrolizumab at approximately 400 mg every 6 weeks to treat TMB-H cancer. In some implementations, children are given pembrolizumab at approximately 2 mg/kg (up to 200 mg) every 3 weeks to treat TMB-H cancer. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat cutaneous squamous cell carcinoma (cSCC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat cSCC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, pembrolizumab is administered at approximately 200 mg every 3 weeks to treat triple-negative breast cancer (TNBC). In some implementations, pembrolizumab is administered at approximately 400 mg every 6 weeks to treat TNBC. In some implementations, pembrolizumab administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration is initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, if the patient or subject is an adult, i.e., for the treatment of an adult indication, an additional dosing regimen of 400 mg every 6 weeks may be used. In some implementations, pembrolizumab administration may be initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some implementations, pembrolizumab administration may be initiated 1, 2, or 3 days after IL-2 administration. In some implementations, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 clone J43 (catalog number BE0033-2) and RMP1-14 (catalog number BE0146) (Bio XCell, West Lebanon, New Hampshire, USA). Many commercially available anti-PD-1 antibodies are known to those skilled in the art.

In some embodiments, the PD-1 inhibitor is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publications Nos. 2010/0266617A1, 2013/0108651A1, and 2013/0109843A2, the disclosures of which are incorporated herein by reference. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody described in U.S. Patent Nos. 8,287,856, 8,580,247, and 8,168,757 and U.S. Patent Application Publications Nos. 2009/0028857A1, 2010/0285013A1, 2013/0022600A1, and 2011/0008369A1, the teachings of which are hereby incorporated by reference. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in U.S. Patent No. 8,735,553B1, the disclosure of which is incorporated herein by reference. In some embodiments, the PD-1 inhibitor is pilithumab, also known as CT-011, described in U.S. Patent No. 8,686,119, the disclosure of which is incorporated herein by reference.

In some embodiments, the PD-1 inhibitor may be a small molecule or a peptide or peptide derivative, such as those described in U.S. Patent Nos. 8,907,053, 9,096,642, and 9,044,442, and U.S. Patent Application Publication No. US2015/0087581; 1,2,4-oxadiazole compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2015/0073024; cyclic peptide mimics and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042; cyclic compounds and derivatives, such as those described in U.S. Patent Application Publication No. US2015/0073042. Those described in US2015/0125491; 1,3,4-oxadiazole and 1,3,4-thiadiazole compounds and derivatives, such as those described in International Patent Application Publication WO 2015/033301; peptide-based compounds and derivatives, such as those described in International Patent Application Publications WO 2015/036927 and WO 2015/04490; or peptide-based macrocyclic compounds and derivatives, such as those described in US Patent Application Publication US2014/0294898; the disclosure of each of these patents is hereby incorporated by reference in its entirety. In some embodiments, the PD-1 inhibitor is cemiplimab, which is available from Regeneron Corporation.

In some embodiments, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors, antagonists, or blockers described in more detail below. Regarding PD-L1 and PD-L2 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or biosimilar. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor may also refer to the compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

In some embodiments, the compositions, processes, and methods described herein include PD-L1 or PD-L2 inhibitors. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof, including its Fab fragment or single-chain variable fragment (scFv). In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2. In some embodiments, the antibody competitively binds to PD-L1 or PD-L2 and/or binds to epitopes on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selective for PD-L1 because the concentration at which the compound binds to or interacts with PD-L1 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L2 receptor. In some embodiments, the compound binds to the PD-L1 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L2 receptor.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2 because the concentration at which the compound binds to or interacts with PD-L2 is much lower than the concentration at which it binds to or interacts with other receptors, including the PD-L1 receptor. In some embodiments, the compound binds to the PD-L2 receptor at a binding constant that is at least about 2 times higher, about 3 times higher, about 5 times higher, about 10 times higher, about 20 times higher, about 30 times higher, about 50 times higher, about 100 times higher, about 200 times higher, about 300 times higher, or about 500 times higher than the concentration bound to the PD-L1 receptor.

Unbound by any theory, it is believed that tumor cells express PD-L1, and T cells express PD-1. However, tumor cell expression of PD-L1 is not required for the efficacy of PD-1 or PD-L1 inhibitors or blockers. In some embodiments, tumor cells express PD-L1. In other embodiments, tumor cells do not express PD-L1. In some embodiments, the method may include a combination of PD-1 and PD-L1 antibodies (e.g., the PD-1 and PD-L1 antibodies described herein) with TIL. The combination of PD-1 and PD-L1 antibodies with TIL may be administered simultaneously or sequentially.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that binds to human PD-L1 and/or PD-L2 with a KD of about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, or about 30 pM or less.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that _{bind to human PD-L1 and/or PD-L2 at a k-association rate of} about ^7.5 × 10⁵ ¹ /M·s or faster, at about 8 × 10⁵ 1/M·s or _faster , at about 8.5 × 10⁵ ¹ /M·s or faster, at about 9 × ^10⁵ 1/M·s or _faster , at about 9.5 × ^10⁵ 1/M·s or _faster , or at about 1 × ^10⁶ 1/M·s or faster. 1/M·s or faster k- _{associations bind} to human PD-L1 and/or PD-L2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that bind to human PD-L1 or PD-L2 with a k- _{dissociation rate} of about 2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k-dissociation rate of about 2.1 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.2 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.3 × ^10⁻⁵ 1/s or slower, to human PD-1 with a k _{-dissociation rate} of about 2.4 × ^10⁻⁵ 1/s or slower, to human PD-1 with _{a k-dissociation rate} of about 2.5 × ^10⁻⁵ 1/s or slower, and to human PD-1 with a k _{-dissociation rate} of about 2.6 × ^{10⁻⁵ 1/s} or slower. It binds to human PD-1 at a k _{dissociation rate} of 1/s or slower, to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 2.7 × ^10⁻⁵ 1/s or slower, or to human PD-L1 or PD-L2 at a k _{dissociation rate} of approximately 3 × ^10⁻⁵ 1/s or slower.

In some embodiments, the PD-L1 and/or PD-L2 inhibitors are PD-L1 and/or PD-L2 inhibitors that block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or less, with an IC50 of about 9 nM or less, with an IC50 of about 8 nM or less, with an IC50 of about 7 nM or less, and with an IC50 of about 6 nM or less. It can either inhibit the binding of human PD-L1 or human PD-L2 to human PD-1, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower, or block human PD-1 or block the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In some embodiments, the PD-L1 inhibitor is durvalumab, also known as MEDI4736 (available from Medimmune, LLC (a subsidiary of AstraZeneca plc) in Gaithersburg, Maryland), or its antigen-binding fragment, conjugate, or variant. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013/0034559, the disclosure of which is incorporated herein by reference. The clinical efficacy of durvalumab has been described in Page et al., Ann. Rev. Med., 2014, 65, 185-202; Brahmer et al., J. Clin. Oncol. 2014, 32, 5s (Supplement, Abstract 8021); and McDermott et al., Cancer Treatment Rev., 2014, 40, 1056-64. The preparation and characterization of durvalumab are described in U.S. Patent No. 8,779,108, the disclosure of which is incorporated herein by reference. The amino acid sequence of durvalumab is set forth in Table 20. The durvalumab monoclonal antibody comprises disulfide bonds at 22-96, 22”-96”, 23”-89”’, 23”’-89”’, 135”-195”’, 135”’-195”’, 148”-204”, 148”-204”, 215”-224”, 215”’-224”, 230-230”, 233-233”, 265-325”, 265”-325”, 371-429” and 371”-429”’; and N-glycosylation sites at Asn-301” and Asn-301”.

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:178 and the light chain provided in SEQ ID NO:179. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of the durvalumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:180, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:181 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises VH and _{VL regions} , each at least 99% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:182, SEQ ID NO:183, and SEQ ID NO:184, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:185, SEQ ID NO:186, and SEQ ID NO:187, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of anti-PD-L1 approved by a regulatory agency, referencing durvalumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application-for-authorization anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab.

Table 20. Amino acid sequences of PD-L1 inhibitors associated with durvalumab.

In some embodiments, the PD-L1 inhibitor is avelumab, also known as MSB0010718C (available from MerckKGaA/EMD Serono) or its antigen-binding fragment, conjugate, or variant. The preparation and characterization of avelumab are described in U.S. Patent Application Publication No. US2014/0341917 A1, the disclosure of which is incorporated herein by reference. The amino acid sequence of avelumab is set forth in Table 21. Avirumab contains heavy chain intra-chain disulfide bonds (C23-C104) at 22-96, 147-203, 264-324, 370-428, 22”-96”, 147”-203”, 264”-324”, and 370”-428”; and light chain intra-chain disulfide bonds (C23-C104) at 22’-90”, 138’-197”, 22”’-90”’, and 138”’-197”’; 223- Intra-heavy disulfide bonds between the heavy and light chains at 215' and 223"-215"' (h 5-CL 126); intra-heavy disulfide bonds between the heavy and light chains at 229"-229" and 232"-232" (h11, h 14); N-glycosylation sites at 300" and 300" (H CH2 N84.4); fucoidan-like CHO polysaccharides of the fucoidan complex; and H CHS K2 C-terminal lysine cleavage at 450" and 450".

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:188 and the light chain provided in SEQ ID NO:189. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of avirumab. In some embodiments, the heavy chain variable region (VH) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:190, and the light chain variable region (VL) of the PD-L1 inhibitor comprises the sequence shown in SEQ ID NO:191 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:195, SEQ ID NO:196, and SEQ ID NO:197, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the PD-L1 inhibitor is a biosimilar monoclonal antibody of an anti-PD-L1 antibody approved by a pharmaceutical regulatory agency, referencing avirumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is avirumab. The anti-PD-L1 antibody may be authorized by a pharmaceutical regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is avelumab.

Table 21. Amino acid sequences of PD-L1 inhibitors associated with avirumab.

In some embodiments, the PD-L1 inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (available from Genentech (a subsidiary of Roche Holding AG) in Basel, Switzerland, under the TECENTRIQ license), or an antigen-binding fragment, conjugate, or variant thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent Application Publications Nos. 2010/0203056A1, 2013/0045200A1, 2013/0045201A1, 2013/0045202A1, or 2014/0065135A1, the disclosure of which is specifically incorporated herein by reference. The preparation and characterization of atezolizumab are described in U.S. Patent No. 8,217,149, the disclosure of which is incorporated herein by reference. The amino acid sequence of atezolizumab is set forth in Table 22. Atezolizumab has intrachain disulfide bonds (C23-C104) at 22-96, 145-201, 262-322, 368-426, 22”-96”, 145”-201”, 262”-322”, and 368”-426”; and at 23”-88”, 134”-194”, 23”’-88”’, and 134”’-194”’. Intra-light chain disulfide bonds (C23-C104); intra-heavy chain disulfide bonds at 221-214' and 221”-214”' (h 5-CL 126); intra-heavy chain disulfide bonds at 227-227” and 230-230” (h 11, h 14); and N-glycosylation sites at 298 and 298' (H CH2 N84.4>A).

In some embodiments, the PD-L1 inhibitor comprises the heavy chain provided in SEQ ID NO:198 and the light chain provided in SEQ ID NO:199. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 96% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain, each being at least 95% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of atezolizumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO:200, and the PD-L1 inhibitor light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO:201 or a conserved amino acid substitution thereof. In some embodiments, the PD-L1 inhibitor comprises _VH and VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, _respectively . In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:204, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:205, SEQ ID NO:206, and SEQ ID NO:207, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to an epitope on PD-L1 identical to any of the aforementioned antibodies.

In some embodiments, the anti-PD-L1 antibody is a biosimilar monoclonal antibody of atezolizumab approved by a regulatory agency. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or patent-pending anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. The anti-PD-L1 antibody may be authorized by a regulatory agency such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab.

Table 22. Amino acid sequences of PD-L1 inhibitors associated with atezolizumab.

In some embodiments, the PD-L1 inhibitor includes those antibodies described in U.S. Patent Application Publication No. 2014/0341917 A1, the disclosure of which is incorporated herein by reference. In other embodiments, an antibody competing with any of these antibodies for binding to PD-L1 is also included. In some embodiments, the anti-PD-L1 antibody is MDX-1105, also known as BMS-935559, disclosed in U.S. Patent No. 7,943,743, the disclosure of which is incorporated herein by reference. In some embodiments, the anti-PD-L1 antibody is selected from the anti-PD-L1 antibody disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference.

In some embodiments, the PD-L1 inhibitor is a commercially available monoclonal antibody, such as INVIVOMAB anti-m-PD-L1 clone 10F.9G2 (catalog number BE0101, Bio X Cell, West Lebanon, New Hampshire, USA). In some embodiments, the anti-PD-L1 antibody is a commercially available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). Many commercially available anti-PD-L1 antibodies are known to those skilled in the art.

In some implementations, the PD-L2 inhibitor is a commercially available monoclonal antibody, such as BIOLEGEND24F.10C12 mouse IgG2a, κ isotype (catalog number 329602, Biolegend, San Diego, California), SIGMA anti-PD-L2 antibody (catalog number SAB3500395, Sigma-Aldrich, St. Louis, Missouri), or other commercially available anti-PD-L2 antibodies known to those skilled in the art.

2. Combination with CTLA-4 inhibitors

In some implementations, TIL therapy provided to cancer patients may include treatment with a therapeutic TIL population alone, or may include combination therapy, including TIL and one or more CTLA-4 inhibitors.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily and is expressed on the surface of helper T cells. CTLA-4 is a negative regulator of CD28-dependent T cell activation and acts as a checkpoint for adaptive immune responses. Similar to the T cell costimulatory protein CD28, CTLA-4 binds to antigens and presents CD80 and CD86 on cells. CTLA-4 delivers inhibitory signals to T cells, while CD28 delivers stimulatory signals. Human antibodies against human CTLA-4 have been described as immunomodulators for many disease conditions, such as for the treatment or prevention of viral and bacterial infections and for the treatment of cancer (WO 01/14424 and WO 00/37504). Several fully human anti-human CTLA-4 monoclonal antibodies (mAbs) have been investigated in clinical trials for the treatment of various types of solid tumors, including (but not limited to) ipilimumab (MDX-010) and trimemumab (CP-675,206).

In some embodiments, the CTLA-4 inhibitor may be any CTLA-4 inhibitor or CTLA-4 blocker known in the art. In particular, it is one of the CTLA-4 inhibitors or blockers described in more detail below. Regarding CTLA-4 inhibitors, the terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor as an antibody may refer to the compound or its antigen-binding fragment, variant, conjugate, or bioanalyte. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor may also refer to a small molecule compound or its pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug.

CTLA-4 inhibitors suitable for the methods of the present invention include (but are not limited to) anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (ipilimumab), trimemumab, anti-CD28 antibodies, anti-CTLA-4 adenettin, anti-CTLA-4 domain antibodies, and single-chain anti-CTLs. The following are listed: A-4 fragment, heavy chain anti-CTLA-4 fragment, light chain anti-CTLA-4 fragment, CTLA-4 inhibitor activating the co-stimulatory pathway, antibody disclosed in PCT Publication No. WO 2001/014424, antibody disclosed in PCT Publication No. WO 2004/035607, antibody disclosed in U.S. Publication No. 2005/0201994, and antibody disclosed in European Patent No. EP 1212422 B1, the disclosures of each of which are incorporated herein by reference. Other CTLA-4 antibodies are described in U.S. Patents 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publications WO 01/14424 and WO 00/37504; and U.S. Publications 2002/0039581 and 2002/086014, the contents of each of which are incorporated herein by reference. Other anti-CTLA-4 antibodies that may be used in the methods of this invention include, for example, antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (Antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998); and U.S. Patents 5,977,318, 6,682,736, 7,109,003 and 7,132,281, the disclosure of each of which is incorporated herein by reference.

Other CTLA-4 inhibitors include (but are not limited to) the following: those that, by means of activation, disrupt the ability of CD28 antigen to bind to its homologous ligand, inhibit the ability of CTLA-4 to bind to its homologous ligand, enhance T cell responses via the co-stimulatory pathway, disrupt the ability of B7 to bind to CD28 and/or CTLA-4, disrupt the ability of B7 to activate the co-stimulatory pathway, disrupt the ability of CD80 to bind to CD28 and/or CTLA-4, disrupt the ability of CD80 to activate the co-stimulatory pathway, disrupt the ability of CD86 to bind to CD28 and/or CTLA-4, disrupt the ability of CD86 to activate the co-stimulatory pathway, and any inhibitor that disrupts the co-stimulatory pathway. This must include: small molecule inhibitors of CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antibodies against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; antisense molecules against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; adenettin against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; RNAi inhibitors (single-stranded and double-stranded) against CD28, CD80, CD86, CTLA-4, and other members of the co-stimulatory pathway; and other CTLA-4 inhibitors.

In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 at a K d , which is about 10  -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, for example, between 10 -13 M and 10 -16 M, or within any range with any two of the foregoing values as endpoints. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is no more than 10 times that of the Kd of ipilimumab. In some embodiments, when compared using the same assay, the Kd of the CTLA-4 inhibitor binding to CTLA-4 is substantially the same as or less than that of ipilimumab (e.g., up to 10 times or up to 100 times lower). In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is no more than 10-fold higher than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively. In some embodiments, when compared using the same assay, the IC50 value of CTLA-4 inhibitors inhibiting the binding of CTLA-4 to CD80 or CD86 is approximately the same as or less than (e.g., up to 10-fold or up to 100-fold lower) than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively.

In some embodiments, a CTLA-4 inhibitor is used in an amount sufficient to inhibit CTLA-4 expression and/or reduce the biological activity of CTLA-4 by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100%. In some embodiments, a CTLA-4 pathway inhibitor is used in an amount sufficient to reduce the biological activity of CTLA-4 by reducing the binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, for example, between 50% and 75%, 75% and 90%, or 90% and 100% relative to a suitable control. In the context of assessing or quantifying the effect of an agent of interest, a suitable control is typically a biological system (e.g., cells or a subject) that has not been exposed to the agent of interest (e.g., a CTLA-4 pathway inhibitor) or has been treated with the agent (or has been exposed to or treated with a negligible amount). In some embodiments, the biological system may serve as its own control, for example, by assessing the biological system before exposure to or treatment with the agent and comparing its status after the start or end of exposure or treatment. In some embodiments, a historical control may be used.

In some embodiments, the CTLA-4 inhibitor is ipilimumab (available from Bristol-Myers Squibb at Yervoy) or its biosimilars, antigen-binding fragments, conjugates, or variants. As known in the art, ipilimumab refers to an anti-CTLA-4 antibody, a fully human IgG 1κ antibody derived from transgenic mice having human genes encoding both heavy and light chains to produce a functional human lineage. Ipilimumab is also mentioned in its CAS Registry No. 477202-00-9 and in PCT Publication No. WO 01/14424, which is incorporated herein by reference in its entirety for all purposes. It is disclosed in the form of Antibody 10DI. Specifically, ipilimumab contains a light chain variable region and a heavy chain variable region (having a light chain variable region comprising SEQ ID NO: 211 and a heavy chain variable region comprising SEQ ID NO: 210). Pharmaceutical compositions of ipilimumab comprise all pharmaceutically acceptable compositions containing ipilimumab and one or more diluents, mediators, or excipients. Examples of pharmaceutical compositions containing ipilimumab are described in International Patent Application Publication No. WO 2007/67959. Ipilimumab can be administered intravenously (IV).

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:208 and the light chain provided in SEQ ID NO:209. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of ipilimumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:210, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:211 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214, or their conserved amino acid substitutions; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:215, SEQ ID NO:216, and SEQ ID NO:217, or their conserved amino acid substitutions. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to ipilimumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of ipilimumab is illustrated in Table 23. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is ipilimumab.

Table 23. Amino acid sequence of ipilimumab.

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, ipilimumab administration may be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, ipilimumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab is administered at approximately mg/kg every 3 weeks for up to 4 consecutive doses to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered as adjuvant therapy for melanoma. In some implementations, ipilimumab is administered at approximately 10 mg/kg every 3 weeks for four doses, followed by 10 mg/kg every 12 weeks for up to 3 years as adjuvant therapy for melanoma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat advanced renal cell carcinoma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 3 weeks, followed by nivolumab at 3 mg/kg on the same day, for a total of 4 doses to treat advanced renal cell carcinoma. In some implementations, after completing the combined 4-dose regimen, nivolumab can be administered as a single agent against advanced renal cell carcinoma and/or renal cell carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, ipilimumab is administered intravenously at approximately 1 mg/kg every 30 minutes over 3 weeks, followed immediately by nivolumab intravenously at 3 mg/kg every 30 minutes on the same day, for a total of 4 doses to treat microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, after completing 4 doses of the composition, nivolumab is administered as a single agent for microsatellite high instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, as recommended according to standard dosing regimens. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also begin 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat hepatocellular carcinoma. In some implementations, ipilimumab is administered intravenously at approximately 3 mg/kg every 3 weeks over 30 minutes, followed immediately by nivolumab intravenously at 1 mg/kg over 30 minutes on the same day, for a total of four doses to treat hepatocellular carcinoma. In some implementations, after completing the combined four-dose regimen, nivolumab is administered as a single agent against hepatocellular carcinoma according to a standard dosing regimen. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, followed by nivolumab at 3 mg/kg every 2 weeks to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks, plus 360 mg nivolumab every 3 weeks for 2 cycles of platinum-based double chemotherapy to treat metastatic non-small cell lung cancer. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some implementations, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, ipilimumab is administered to treat malignant pleural mesothelioma. In some implementations, ipilimumab is administered at approximately 1 mg/kg every 6 weeks and nivolumab at 360 mg every 3 weeks to treat malignant pleural mesothelioma. In some implementations, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

Trimelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has CAS number 745013-59-6. Trimelimumab is disclosed in U.S. Patent No. 6,682,736 (incorporated herein by reference) as antibody 11.2.1. The amino acid sequences of the heavy and light chains of trimemumab are described in SEQ IND NO: 218 and 219, respectively. Trimelimumab has been studied in clinical trials for the treatment of various tumors, including melanoma and breast cancer; wherein it is administered intravenously in single or multiple doses at a dose range between 0.01 and 15 mg/kg every 4 or 12 weeks. In the embodiments provided by the present invention, trimemumab is administered topically, particularly intradermally or subcutaneously. The effective amount of trimemumab administered intradermally or subcutaneously is typically in the range of 5-200 mg/dose per person. In some implementations, the effective dose of trimemumab is in the range of 10-150 mg/dose per person. In some specific implementations, the effective dose of trimemumab is approximately 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175 or 200 mg/dose per person.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:218 and the light chain provided in SEQ ID NO:219. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of trimexumab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:220, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:221 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in _SEQ ID NO:220 and _SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:225, SEQ ID NO:226, and SEQ ID NO:227, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a biosimilar monoclonal antibody of anti-CTLA-4 approved by a regulatory agency, referencing trimemumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody having at least 97% sequence identity with an amino acid sequence of a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and comprising one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of trimemumab is illustrated in Table 24. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is trimemumab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is trimexumab.

Table 24. Amino acid sequence of trimemumab.

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, wherein trimemumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered at doses of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, trimemumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, trimemumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some implementations, the CTLA-4 inhibitor is trimemumab or a biosimilar thereof, and trimemumab is administered every 2, 3, 4, 5, or 6 weeks. In some implementations, trimemumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is zeflimab from Agenus or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Zeflimab is a fully human monoclonal antibody. Zeflimab is designated Chemical Abstracts Service (CAS) Registry No. 2148321-69-9 and is also known as AGEN1884. The preparation and characterization of zeflimab are described in U.S. Patent No. 10,144,779 and U.S. Patent Application Publication No. US2020/0024350A1, the disclosures of which are incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor comprises the heavy chain provided in SEQ ID NO:228 and the light chain provided in SEQ ID NO:229. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 99% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 98% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain each being at least 97% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 96% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain, each at least 95% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of zeflimab. In some embodiments, the heavy chain variable region ( _VH ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:230, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor comprises the sequence shown in SEQ ID NO:231 or a conserved amino acid substitution thereof. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 99% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 98% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises _VH and _VL regions, each at least 97% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 96% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region, each being at least 95% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:231, SEQ ID NO:233, and SEQ ID NO:234, or their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:235, SEQ ID NO:236, and SEQ ID NO:237, or their conserved amino acid substitutions, respectively. In some embodiments, the antibody competitively binds to and/or binds to the epitopes on CTLA-4 identical to those of any of the aforementioned antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a regulatory agency with reference to zeflimab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody containing an amino acid sequence having at least 97% sequence identity with a reference drug or reference biological product, such as 97%, 98%, 99%, or 100% sequence identity, and containing one or more post-translational modifications relative to the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of zeflimab is illustrated in Table 25. In some embodiments, the biosimilar is an authorized or patent-pending anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the reference drug or reference biological product is zeflimab. Anti-CTLA-4 antibodies are eligible for authorization from regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein said one or more excipients are the same as or different from those contained in a reference drug or reference biological product, wherein said reference drug or reference biological product is zeflimab.

Table 25. Amino acid sequence of zefrizinumab.

Other examples of anti-CTLA-4 antibodies include (but are not limited to): AAGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659, and ADG116, which are known to those skilled in the art.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent disclosures: US2019/0048096 A1; US2020/0223907; US2019/0201334; US2019/0201334; US2005/0201994; EP 1212422B1; WO 2018/204760; WO 2018/204760; WO 2001/014424; WO2 WO 2004/035607; WO 2003/086459; WO 2012/120125; WO 2000/037504; WO 2009/100140; WO2006/09649; WO2005092380; WO 2007/123737; WO 2006/029219; WO 2010/0979597; WO2006/12168; and WO1997020574, each of which is incorporated herein by reference. Other CTLA-4 antibodies are described in the following: U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227 and 6,984,720; PCT Publications Nos. WO 01/14424 and WO 00/37504; and U.S. Publications Nos. 2002/0039581 and 2002/086014; and/or U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281, each of which is incorporated herein by reference. In some embodiments, the anti-CTLA-4 antibody is, for example, one of the antibodies disclosed in: WO 98/42752; U.S. Patents 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 10067-10071 (1998); Camacho et al., J. Clin. Oncol., 2004, 22, 145 (Abstract No. 2505 (2004) (Antibody CP-675206); or Mokyr et al., Cancer Res., 1998, 58, 5301-5304 (1998), each of which is incorporated herein by reference.

In some implementations, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996/040915 (incorporated herein by reference).

In some implementations, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, anti-CTLA-4 RNAi molecules may be in the form described in: PCT Publications WO 1999/032619 and WO 2001/029058; U.S. Publications 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, and 2005/01009. Patent Nos. 13, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443; and/or U.S. Patent Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in European Patent No. 1309726 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule as described in U.S. Patent Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In some embodiments, the CTLA-4 inhibitor is an aptamer described in International Patent Application Publication No. WO2004/081021 (incorporated herein by reference).

In other embodiments, the anti-CTLA-4 RNAi molecule of the present invention is the RNA molecule described in U.S. Patent Nos. 5,898,031, 6,107,094, 7,432,249 and 7,432,250 and European Application No. EP 0928290 (incorporated herein by reference).

3. Preconditioning of lymphocyte depletion in patients

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the present disclosure. In some embodiments, the present invention includes a TIL population for treating cancer in patients pretreated with non-myeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/ ^m² /day for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, following the non-myeloablative chemotherapy and TIL infusion (day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 (aldeleukin, available from PROLEUKIN) every 8 hours at 720,000 IU/kg to achieve physiological tolerance. In some implementations, TIL groups are used in combination with IL-2 to treat cancer, wherein IL-2 is administered after the TIL group.

Experimental findings have shown that lymphocyte depletion plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system ('cytokine pool') prior to adoptive metastasis of tumor-specific T lymphocytes. Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as "immunosuppressive modulation") in a patient prior to the introduction of the TIL of the present invention.

Generally, lymphocyte depletion is achieved by administration of fludarabine or cyclophosphamide (the active form of which is called equiphosphamide) and combinations thereof. Such methods are described in Gassner et al., Cancer Immunol. Immunother. 2011, 60, 75-85; Muranski et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-5239; and Dudley et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL. In some embodiments, fludarabine is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, fludarabine is administered at doses of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2–7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4–5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4–5 days.

In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, cyclophosphamide is administered to obtain the active form of cyclophosphamide, horsephosphamide, at a concentration of 1 μg/mL. In some embodiments, cyclophosphamide is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, cyclophosphamide is administered at doses of 100 mg/m²/day, 150 mg/m²/day, 175 mg/m²/day, 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day, 275 mg/m²/day, or 300 mg/m²/day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.e., i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2–7 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4-5 days. In some implementations, cyclophosphamide treatment is administered intravenously at 250 mg/m²/day for 4 days.

In some implementations, lymphocyte depletion is achieved by administering fludarabine and cyclophosphamide together to the patient. In some implementations, fludarabine is administered intravenously at 25 mg/m²/day for 4 days, and cyclophosphamide is administered intravenously at 250 mg/m²/day.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administering fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day for two days and fludarabine at a dose of 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered for the first two days and lymphocyte depletion is achieved over a total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 25 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 50 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 20 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of about 40 mg/m²/day for two days and fludarabine at a dose of about 15 mg/m²/day for five days, wherein both cyclophosphamide and fludarabine are administered in the first two days and lymphocyte depletion is achieved in the total of five days.

In some implementations, lymphocyte depletion is achieved by administering cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by fludarabine at a dose of 25 mg/m²/day for three days.

In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, mesna is administered at a dose of 15 mg/kg. In some embodiments, mesna is infused, and if continuous infusion is initiated over 24 hours, with the respective cyclophosphamide dose starting, mesna may be infused with cyclophosphamide over approximately 2 hours (days -5 and/or -4), and then infused at a rate of 3 mg/kg/hour for the remaining 22 hours.

In some implementations, lymphocyte depletion includes the following steps: starting the day after administering the third TIL group to the patient, treating the patient with an IL-2 regimen.

In some implementations, lymphocyte depletion includes the following steps: starting on the day the third TIL group is administered to the patient, the patient is treated with an IL-2 regimen.

In some embodiments, lymphocyte depletion comprises a 5-day preconditioning treatment. In some embodiments, the days are indicated as day-5 to day-1, or day 0 to day 4. In some embodiments, the regimen comprises cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, the regimen comprises 60 mg/kg intravenous cyclophosphamide on days-5 and-4 (i.e., day 0 and day 1). In some embodiments, cyclophosphamide is administered in combination with mesna. In some embodiments, the regimen also comprises fludarabine. In some embodiments, the regimen also comprises intravenous fludarabine. In some embodiments, the regimen also comprises 25 mg/ ^m² intravenous fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine at a dose of 25 mg/ ^m² on days -5 and ^-1 (i.e., days 0 to 4).

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for five days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for one day.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the following steps: administration of cyclophosphamide at a dose of 60 mg/m²/day and fludarabine at a dose of 25 mg/m²/day for two days, followed by administration of fludarabine at a dose of 25 mg/m²/day for three days.

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 26.

Table 26. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 27.

Table 27. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 28.

Table 28. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 25 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 29.

Table 29. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25 mg/m²/day			X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 30.

Table 30. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day	X	X	X	X	X
TIL infusion						X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 31.

Table 31. Exemplary lymphocyte depletion and treatment regimens.

sky

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 32.

Table 32. Exemplary lymphocyte depletion and treatment regimens.

sky

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300 mg/kg

X

X

Mesna (as needed)

X

X

Fludarabine 30 mg/m²/day

X

X

X

TIL infusion

X

In some implementations, the non-myeloablative lymphocyte depletion regimen is administered according to Table 33.

Table 33. Exemplary lymphocyte depletion and treatment regimens.

sky	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300 mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30 mg/m²/day			X	X	X
TIL infusion						X

In some embodiments, the TIL infusion used in conjunction with the above-described myeloablative lymphocyte depletion regimens may be any TIL composition described herein, as well as an IL-2 addition regimen and administration of combination therapies as described herein (e.g., PD-1 and PD-L1 inhibitors).

4. IL-2 scheme

In some implementations, the IL-2 regimen includes a high-dose IL-2 regimen comprising alephrine or a biosimilar or variant thereof, administered intravenously starting the day after the effective fraction of treatment in the therapeutic TIL population. Alephrine or a biosimilar or variant thereof is administered as a 15-minute bolus intravenous infusion every eight hours at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient weight) until tolerated, for a maximum of 14 doses. After a 9-day rest period, this course can be repeated for another 14 doses, for a maximum total of 28 doses. In some implementations, IL-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In some implementations, IL-2 is administered at a maximum dose of up to 6 doses.

In some implementations, the IL-2 regimen comprises a tapering IL-2 regimen. Tapering IL-2 regimens have been described in O'Day et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In some implementations, tapering IL-2 therapy comprises an intravenous administration of 18 × ^10⁶ IU/ ^m² over 6 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 12 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 24 hours, followed by an intravenous administration of 4.5 × ^10⁶ IU/ ^m² of adeleukin or a biosimilar or variant thereof over 72 hours. This treatment cycle may be repeated every 28 days for a maximum of four cycles. In some implementations, the decreasing IL-2 regimen includes 18,000,000 IU/ ^m² on day 1, 9,000,000 IU/ ^m² on day 2, and 4,500,000 IU/ ^m² on days 3 and 4.

In some implementations, the IL-2 regimen includes a low-dose IL-2 regimen. Any low-dose IL-2 regimen known in the art may be used, including those described in Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann et al., Lancet Diabetes Endocrinol. 2013, 1, 295-305; and Rosenzwaig et al., Ann. Rheum. Dis. 2019, 78, 209-217, the disclosures of which are incorporated herein by reference. In some implementations, a low-dose IL-2 regimen comprises 18 × ^10⁶ IU/ ^m² of interleukin or its biosimilar or variant every 24 hours, administered as a continuous infusion for 5 days; followed by a 2–6 day period without IL-2 therapy; optionally followed by intravenous administration of interleukin or its biosimilar or variant at 18 × ^10⁶ IU/ ^m² every 24 hours for another 5 days; optionally followed by a 3-week period without IL-2 therapy, after which other cycles may be administered.

In some implementations, IL-2 is administered at a maximum dose of up to 6 doses. In some implementations, high-dose IL-2 regimens are modified for pediatric use. In some implementations, interleukin is administered at a dose of 600,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 400,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 500,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 300,000 IU/kg every 8-12 hours for up to 6 doses. In some implementations, interleukin is administered at a dose of 200,000 IU/kg every 8-12 hours, for a maximum of 6 doses. In some implementations, interleukin is administered at a dose of 100,000 IU/kg every 8-12 hours, for a maximum of 6 doses.

In some embodiments, the IL-2 regimen comprises administering PEGylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bepedipine interleukin or a fragment, variant, or bioanalyte thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen comprises administering THOR-707 or a fragment, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some implementations, the IL-2 regimen includes administration of Nevorin Alpha or a fragment, variant, or biosimilar thereof following TIL administration. In some implementations, Nevorin Alpha is administered to the patient at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 protocol includes administering an IL-2 fragment grafted onto the antibody backbone. In some embodiments, the IL-2 protocol includes administering an antibody cytokine graft protein that binds to a low-affinity IL-2 receptor. In some embodiments, the antibody cytokine graft protein includes a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof grafted into the CDR of _VH or _VL , wherein the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine transplantation protein comprises a heavy chain variable region ( _VH ) containing complementarity-determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) containing LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof, which is transplanted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody cytokine transplantation protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody or a fragment thereof, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days, wherein the antibody comprises a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

In some implementations, the serum half-life of the antibody cytokine transplanted protein described herein is longer than that of wild-type IL-2 molecules (e.g., but not limited to interleukin or comparable molecules).

In some embodiments, the TIL infusion used in conjunction with the aforementioned embodiments of the myeloablative lymphocyte depletion regimen may be any TIL composition described herein and may also include MIL and PBL infusions in lieu of TIL infusion, as well as the addition of an IL-2 regimen and administration of combination therapies as described herein (e.g., PD-1 and/or PD-L1 inhibitors and/or CTLA-4 inhibitors).

Implementation Plan

This article provides methods for amplifying TILs and generating therapeutic TIL populations, the methods including methods for gene editing at least a portion of TILs to enhance their therapeutic efficacy.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating the fourth TIL population takes about 9 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 bead agonist or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about one day.

In some implementations, the process of cultivating the fourth TIL population takes about 2 days.

In some implementations, the process of cultivating a fourth TIL population takes about 3 days.

In some implementations, the process of cultivating the fourth TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing a third TIL population takes about 8 days.

In some implementations, the process of culturing a third TIL population takes approximately 9 days.

In some implementations, the process of culturing the third TIL population takes about 8-9 days.

In some implementations, the process of cultivating a third TIL population takes about 10 days.

In some implementations, the process of culturing a third TIL population takes approximately 8-10 days.

In some implementations, the process of culturing a third TIL population takes approximately 11 days.

In some implementations, the process of culturing a third TIL population takes approximately 12 days.

In some implementations, the process of cultivating a third TIL population takes approximately 13 days.

In some implementations, the process of culturing a third TIL population takes approximately 14 days.

In some implementations, the process of cultivating a third TIL population takes about 15 days.

In some implementations, the step of culturing a third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating the first TIL population takes about 3 days.

In some implementations, the process of cultivating the first TIL population takes about 4 days.

In some implementations, the process of cultivating the first TIL population takes about 5 days.

In some implementations, the process of culturing the first TIL population takes about 6 days.

In some implementations, the process of cultivating the first TIL population takes about 7 days.

In some implementations, the process of cultivating the first TIL population takes about 8 days.

In some implementations, the process of cultivating the first TIL population takes about 9 days.

In some implementations, the process of cultivating a third TIL population takes about one day.

In some implementations, the process of cultivating a third TIL population takes about 2 days.

In some implementations, the process of cultivating a third TIL population takes about 3 days.

In some implementations, the process of cultivating a third TIL population takes about 4 days.

In some implementations, the process of cultivating a third TIL population takes about 5 days.

In some implementations, the process of culturing a third TIL population takes about 6 days.

In some implementations, the process of culturing a third TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) Culture the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) A fourth population of TILs was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(f) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the process of cultivating the fourth TIL population takes about 5 days.

In some implementations, the process of cultivating a fourth TIL population takes about 6 days.

In some implementations, the process of cultivating the fourth TIL population takes about 7 days.

In some implementations, the process of cultivating the fourth TIL population takes about 8 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately nine days.

In some implementations, the process of culturing the fourth TIL population takes about 8-9 days.

In some implementations, the process of cultivating the fourth TIL population takes about 10 days.

In some implementations, the process of culturing the fourth TIL population takes about 8-10 days.

In some implementations, the process of cultivating the fourth TIL population takes about 11 days.

In some implementations, the process of cultivating the fourth TIL population takes about 12 days.

In some implementations, the process of cultivating the fourth TIL population takes about 13 days.

In some implementations, the process of cultivating a fourth TIL population takes approximately 14 days.

In some implementations, the process of cultivating the fourth TIL population takes about 15 days.

In some implementations, the step of culturing a fourth TIL population is performed by: culturing the fourth TIL population in a third cell culture medium for a first period of approximately 1-7 days; at the end of the first period, splitting the culture into multiple subcultures; culturing each of the multiple subcultures in a fourth culture medium containing IL-2 for a second period of approximately 3-7 days; and at the end of the second period, merging the multiple subcultures to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, the second culture period is approximately 7 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(f) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digest the tumor tissue in an enzyme medium to produce tumor digestate;

(c) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(d) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to generate a third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(g) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the process of cultivating a second TIL population takes about 2 days.

In some implementations, the process of cultivating a second TIL population takes about 3 days.

In some implementations, the process of cultivating a second TIL population takes about 4 days.

In some implementations, the step of culturing a third or fourth TIL population takes about one day.

In some implementations, the process of culturing a third or fourth TIL population takes about 2 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 3 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 4 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 5 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 6 days.

In some implementations, the process of culturing a third or fourth TIL population takes about 7 days.

In some implementations, the process of culturing multiple subcultures takes about 3 days.

In some implementations, the process of culturing multiple subcultures takes about 4 days.

In some implementations, the process of culturing multiple subcultures takes about 5 days.

In some implementations, the process of culturing multiple subcultures takes about 6 days.

In some implementations, the process of culturing multiple subcultures takes about 7 days.

In some implementations, during the step of culturing a first TIL population in a first culture medium, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in the first culture medium.

In some embodiments, during the step of culturing a second TIL population in a second culture medium, the second culture medium further contains anti-CD3 and anti-CD28 beads or antibodies.

In some implementations, anti-CD3 and anti-CD28 beads or antibodies contain OKT-3 in a second culture medium.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of a first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Digesting tumor fragments in an enzyme medium to produce tumor digests;

(c) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(d) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(e) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the initiation of the first amplification takes approximately one day.

In some implementations, the initiation of the first amplification takes approximately 2 days.

In some implementations, the initiation of the first amplification takes approximately 3 days.

In some implementations, the initiation of the first amplification takes approximately 4 days.

In some implementations, the initiation of the first amplification takes approximately 5 days.

In some implementations, the initiation of the first amplification takes approximately 6 days.

In some implementations, the initiation of the first amplification takes approximately 7 days.

In some implementations, the initiation of the first amplification takes approximately 8 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the rapid second amplification takes approximately 9 days.

In some implementations, the rapid second amplification takes approximately 10 days.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the first culture period is approximately one day.

In some implementations, the first culture period is approximately 2 days.

In some implementations, the first culture period is approximately 3 days.

In some implementations, the first culture period is approximately 4 days.

In some implementations, the first culture period is approximately 5 days.

In some implementations, the first culture period is approximately 6 days.

In some implementations, the first culture period is approximately 7 days.

In some implementations, the second culture period is approximately 3 days.

In some implementations, the second culture period is approximately 4 days.

In some implementations, the second culture period is approximately 5 days.

In some implementations, the second culture period is approximately 6 days.

In some implementations, all steps are completed within a period of approximately 16-18 days.

In some implementations, all steps are completed within a period of approximately 16 days.

In some implementations, all steps are completed within a period of approximately 17 days.

In some implementations, all steps are completed within a period of approximately 18 days.

In some implementations, all steps are completed within a period of approximately 18-22 days.

In some implementations, all steps are completed within a period of approximately 19 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 20 days.

In some implementations, all steps are completed within a period of approximately 21 days.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, the first culture medium contains APC.

In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

In some embodiments, the first culture medium contains OKT-3.

In some implementations, the first culture medium further contains anti-CD3 and anti-CD28 beads or antibodies during the initial amplification step.

In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise OKT-3.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

In some implementations, this document provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) Digesting tumor tissue samples in an enzyme medium to produce tumor digests;

(c) The tumor digest is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-9 days to obtain the second TIL population.

(d) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(f) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(g) Collect the therapeutic TIL population obtained from step (f), wherein each of steps (c) to (g) is performed in a closed, sterile system, and wherein the transition from step (c) to step (d), the transition from step (d) to step (e), the transition from step (e) to step (f) and/or the transition from step (f) to step (g) is performed without opening the system.

In some embodiments, the enzyme medium comprises a DNA enzyme.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises a neutral protease.

In some embodiments, the enzyme medium comprises hyaluronidase.

In some implementations, the first amplification takes about 3 days.

In some implementations, the first amplification takes about 4 days.

In some implementations, the first amplification takes about 5 days.

In some implementations, the first amplification takes about 6 days.

In some implementations, the first amplification takes about 7 days.

In some implementations, the first amplification takes about 8 days.

In some implementations, the first amplification takes about 9 days.

In some implementations, the process of activating the second TIL population takes about one day.

In some implementations, the process of activating the second TIL population takes about 2 days.

In some implementations, the process of activating the second TIL population takes about 3 days.

In some implementations, the process of activating the second TIL population takes about 4 days.

In some implementations, the process of activating the second TIL population takes about 5 days.

In some implementations, the process of activating the second TIL population takes about 6 days.

In some implementations, the process of activating the second TIL population takes about 7 days.

In some implementations, the second amplification takes about 5 days.

In some implementations, the second amplification takes about 6 days.

In some implementations, the second amplification takes about 7 days.

In some implementations, the second amplification takes approximately 8 days.

In some implementations, the second amplification takes approximately 9 days.

In some implementations, the second amplification takes about 8-9 days.

In some implementations, the second amplification takes about 10 days.

In some implementations, the second amplification takes about 8-10 days.

In some implementations, the second amplification takes approximately 11 days.

In some implementations, the second amplification takes approximately 12 days.

In some implementations, the second amplification takes approximately 13 days.

In some implementations, the second amplification takes approximately 14 days.

In some implementations, the second amplification takes about 15 days.

In some implementations, the second amplification is performed through the following steps:

(i) The first period of culturing the fourth TIL population in the second culture medium for about 5 days.

(ii) The culture from step (i) is subdivided into multiple subcultures, each of which is transferred to a separate sealed container providing a third breathable surface and cultured for a second period of approximately 4 or 5 days in a third medium containing IL-2, wherein the transition from step (i) to step (ii) is performed without opening the system, and

(iii) Multiple subcultures are merged to produce a fifth TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

In some implementations, all steps are completed within a period of approximately 22 days.

In some implementations, all steps are completed within a period of approximately 19-22 days.

In some implementation schemes, all steps are completed within a period of approximately 19-20 days.

In some implementations, all steps are completed within a period of approximately 20-22 days.

In some implementations, all steps are completed within a period of approximately 23 days.

In some implementations, all steps are completed within a period of approximately 24 days.

In some implementations, all steps are completed within a period of approximately 25 days.

In some implementations, all steps are completed within a period of approximately 26 days.

In some implementations, all steps are completed within a period of approximately 27 days.

In some implementations, all steps are completed within a period of approximately 28 days.

In some implementations, all steps are completed within a period of approximately 29 days.

In some implementations, all steps are completed within a period of approximately 30 days.

In some implementations, all steps are completed within a period of approximately 31 days.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 for about 3–9 days to produce a second TIL population.

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population;

(c) The third TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(d) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Digesting a first TIL population from tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a third cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3 days to generate a second population of TILs;

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) Culture the third TIL population for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of a fourth TIL population; and

(e) The culture of the fourth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining a first TIL population by digesting tumor tissue removed from a subject or patient in an enzyme medium to produce tumor digests;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population;

(c) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(f) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 was cultured for about 3 days to produce a second TIL population.

(b) The second TIL population was cultured in a second cell culture medium containing IL-2 and OKT-3 for 2–4 days to generate a third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(d) The fourth TIL population was cultured for approximately 1–7 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce a culture of the fifth TIL population; and

(e) The culture of the fifth TIL population is split into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for about 3-7 days, and the multiple subcultures are combined to provide a fifth TIL population containing an expanded number of TILs.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a patient or subject in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 and anti-CD28 beads or antibodies to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving a first TIL population by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, this document provides a method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium to produce tumor digests in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 1 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

In some embodiments, rapid second expansion is performed by culturing a third or fourth TIL population in a second cell culture medium for approximately 1-7 days in a first period. At the end of the first period, the culture is split into multiple subcultures, each of which is cultured in a third culture medium containing IL-2 for approximately 3-6 days in a second period. At the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

In some implementations, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

In some implementations, the electroporation step consists of a single electroporation event mediating the transfer of at least two gene editors.

In some implementations, each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via an electroporation event.

In some implementations, the electroporation step further includes a rest period following each electroporation event.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first gene editor to regulate the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to regulate the expression of a second protein, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

In some embodiments, the electroporation step includes a first electroporation event mediating the transfer of a first TALE nuclease system, a first resting period, a second electroporation event mediating the transfer of a second TALE nuclease system, and a second resting period, wherein the first resting period may be the same as or different from the second resting period.

In some implementations, the first resting period and the second resting period comprise incubating a third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the first and second resting periods include incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some implementations, the first rest period and the second rest period are independently about 10 hours to 5 days.

In some implementations, the first rest period and the second rest period are independently approximately 10 hours to 3 days.

In some implementations, the first resting period is approximately 1 to 3 days.

In some implementations, the first rest period is approximately 3 days.

In some implementations, the second resting period is approximately 10 hours to 1 day.

In some implementations, the second resting period is approximately 12 to 24 hours.

In some implementations, the second resting period is approximately 15 hours to approximately 18 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

In some embodiments, the second resting period includes incubating a third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

In some implementations, the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

In some embodiments, the step of gene editing at least a portion of a second or third TIL population includes a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

In some embodiments, the gene editor is a TALE nuclease system for regulating the expression of at least one protein.

In some embodiments, the TALE nuclease system regulates the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and CTLA-4.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of PD-1 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 and TIGIT expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 and CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and CBL-B.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CTLA-4 and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH and LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and LAG-3.

In some implementations, the gene editor includes a TALE nuclease system that regulates the expression of CBL-B and TIGIT.

In some implementations, the gene editor includes a TALE nuclease system that regulates PD-1 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CTLA-4 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CISH expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates CBL-B expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates LAG-3 expression.

In some implementations, the gene editor includes a TALE nuclease system that regulates TIGIT expression.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the quiescent step includes incubating a third or fourth TIL population at about 30-40°C and about 5% _CO2 .

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for about one day after the gene editing step and before the step of culturing the third or fourth TIL population.

In some embodiments, the method further includes a step of quiescent the third or fourth TIL population for approximately 12 to 24 hours after the gene editing step and before the step of culturing the third or fourth TIL population.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 to 18 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes quiescent the third or fourth TIL population for approximately 15 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 23 hours.

In some implementations, the step of quiescent the third or fourth TIL population includes incubating the third or fourth TIL population in a cell culture medium containing IL-2 at 37°C for about one hour, followed by incubation at about 30°C for about 15 to 22 hours.

In some embodiments, the first protein and the second protein are independently selected from PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein is different from the second protein.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

In some implementations, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

In some implementations, the first protein is PD-1 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is PD-1.

In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

In some embodiments, the first protein is TIGIT and the second protein is PD-1.

In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is CTLA-4.

In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

In some embodiments, the first protein is LAG-3 and the second protein is CISH.

In some implementations, the first protein is CISH and the second protein is LAG-3.

In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

In some embodiments, the first protein is CISH and the second protein is CBL-B.

In some embodiments, the first protein is CBL-B and the second protein is CISH.

In some implementations, the first protein or the second protein is PD-1.

In some implementations, the first protein or the second protein is CTLA-4.

In some implementations, the first protein or the second protein is LAG-3.

In some implementations, the first protein or the second protein is CISH.

In some embodiments, the first protein or the second protein is CBL-B.

In some implementations, the first protein or the second protein is TIGIT.

In some implementations, the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

In some implementations, the expanded number of TILs includes a population of therapeutic TILs.

In some implementations, the antigen-presenting cells (APCs) are PBMCs.

In some implementations, the PBMC is irradiated and allogeneic.

In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

In some embodiments, the first cell culture medium and/or the second cell culture medium further contain a 4-1BB agonist and/or an OX40 agonist.

In some implementations, this document provides expanded numbers of tumor-infiltrating lymphocytes (TILs) or therapeutic TIL populations produced by the methods described herein.

In some embodiments, this document provides pharmaceutical compositions comprising an amplified number of TILs or a therapeutic population of TILs as described herein and a pharmaceutically acceptable carrier.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of an expanded number of TILs or a therapeutically active population of TILs as described herein.

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some embodiments, this document provides a method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), which include:

(a) Obtaining a first TIL population from a tumor excised from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor digest to a closed system and performing a first amplification to produce a second TIL population by culturing the first TIL population in a first cell culture medium containing IL-2, wherein the first amplification is performed in a closed container providing a first ventilated surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, CISH, CBL-B, TIGIT, and/or LAG-3.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting PD-1 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CTLA-4 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting CISH expression.

In some implementations, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting LAG-3 expression.

In some implementations, the electroporation step also includes delivery of a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CTLA-4 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting PD-1 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CISH expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CTLA-4 expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting CBL-B expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CISH expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting LAG-3 expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting CBL-B expression and a TALEN system for inhibiting TIGIT expression.

In some embodiments, the electroporation step further includes delivery of a TALEN system for inhibiting LAG-3 expression and a TALEN system for inhibiting TIGIT expression.

In some implementations, the therapeutically effective dose of TIL is approximately 2.3 × 10¹⁰ to approximately 13.7 × 10¹⁰ TILs.

In some implementations, a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of TIL cells in step (k).

In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins the day after the administration of TIL cells to the patient in step (k).

In some implementations, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is metastatic melanoma.

In some implementations, the cancer is NSCLC.

In some implementations, the cancer is metastatic NSCLC.

In some implementations, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Example

The embodiments covered herein are now described with reference to the following examples. These examples are provided for illustrative purposes only and this disclosure should in no way be construed as limiting to these examples, but rather as covering any and all variations that become apparent from the teachings provided herein.

Example 1: Preparation of culture medium for pre-REP and REP processes

This embodiment describes a procedure for preparing a tissue culture medium suitable for cultures involving tumor-infiltrating lymphocytes (TILs) derived from various solid tumors. This culture medium can be used to prepare any of the TILs described in this application and other embodiments.

Prepare CM1 . Remove the following reagents from the freezer and warm them in a 37°C water bath: (RPMI 1640, human AB serum, 200 mM L-glutamine). Prepare CM1 medium by adding each component to the top of a 0.2 μm filter unit suitable for the desired filtration volume, according to Table 34 below. Store at 4°C.

Table 34. Preparation of CM1

On the day of use, preheat the required amount of CM1 in a 37°C water bath and add 6000 IU/mL IL-2.

Additional information may be added as needed, based on Table 35.

Table 35. Additional information for CM1 as needed.

Preparation of CM2

Remove the prepared CM1 from the refrigerator or prepare fresh CM1. Remove AIM- from the refrigerator and prepare the required amount of CM2 by mixing the prepared CM1 with an equal volume of AIM- in a sterile culture medium bottle. On the day of use, add 3000 IU/mL IL-2 to the CM2 culture medium. On the day of use, prepare a sufficient amount of CM2 with 3000 IU/mL IL-2. Label the CM2 culture medium bottle with the name, the initials of the preparer's name, the filtration/preparation date, and the two-week expiry date, and store at 4°C until needed for tissue culture.

Preparation of CM3

Prepare CM3 on the day it is needed. CM3 is the same as AIM-medium, but supplemented with 3000 IU/mL IL-2 on the day of use. Prepare the amount of CM3 needed for the experiment by directly adding the IL-2 stock solution to the AIM-V bottle or bag. Mix thoroughly by gentle shaking. Immediately after adding to AIM-V, label the bottle “3000 IU/mL IL-2”. If there is excess CM3, store it in a bottle at 4°C, labeling it with the medium name, the preparer's initials, the date of preparation, and its expiration date (7 days after preparation). After storing at 4°C for 7 days, discard the medium supplemented with IL-2.

Preparation of CM4

CM4 is the same as CM3, but with an additional 2 mM GlutaMAX ^™ (final concentration). Add 10 mL of 200 mM GlutaMAX ^™ per 1 L of CM3. Prepare the required amount of CM4 by directly adding IL-2 and GlutaMAX ^™ stock solution to the AIM-V bottle or bag. Mix thoroughly with gentle shaking. Immediately after adding to the AIM-V, label the bottle “3000 IL/mL IL-2 and GlutaMAX”. If excess CM4 exists, store it in the bottle at 4°C, labeling it with the culture medium name, “GlutaMAX”, and its expiration date (7 days after preparation). Discard the culture medium supplemented with IL-2 after storage at 4°C for more than 7 days.

Example 2: Use of a mixture of IL-2, IL-15 and IL-21 cytokines

This embodiment describes the use of IL-2, IL-15, and IL-21 cytokines, which act as additional T cell growth factors, in combination with the TIL process of any embodiment herein.

Using the procedures described herein, at the start of culture, TILs were observed to grow from tumors in one experimental group in the presence of IL-2 and in another group in the presence of a combination of IL-2, IL-15, and IL-21, which substituted for IL-2. At the completion of pre-REP, the amplification, phenotype, function (CD107a+ and IFN-γ), and TCR Vβ lineage of the cultures were assessed. IL-15 and IL-21 are described elsewhere in this paper and in Santegoets et al., J. Transl. Med., 2013, 11, 37.

The results showed enhanced TIL amplification (>20%) in CD4 ⁺ and CD8 ⁺ cells under IL-2, IL-15, and IL-21 treatments compared to IL-2-only conditions. Significant skewness towards a CD8 ⁺ population with a skewed TCR Vβ lineage was observed in TILs obtained from IL-2, IL-15, and IL-21-treated cultures compared to IL-2-only cultures. IFN-γ and CD107a levels were increased in TILs treated with IL-2, IL-15, and IL-21 compared to IL-2-only TILs.

Example 3: Quantification of individual batches of γ-irradiated peripheral mononuclear cells

This embodiment describes a simplified procedure for quantifying individual batches of γ-irradiated peripheral monocytes (PBMCs, also known as monocytes or MNCs) used as allogeneic feeder cells in the exemplary methods described herein.

Each batch of irradiated MNC feeder cells was prepared from individual donors. Each batch or donor was individually screened based on its ability to amplify TILs in the REP in the presence of purified anti-CD3 (clonal OKT3) antibody and interleukin-2 (IL-2). Furthermore, each batch of feeder cells was tested without the addition of TILs to verify that the received gamma irradiation dose was sufficient to prevent replication.

TIL regeneration requires γ-irradiated, growth-arrested MNC feeder cells. Membrane receptors on the feeder MNCs bind to anti-CD3 (clonal OKT3) antibodies and cross-link with TILs in the REP culture flask, stimulating TIL expansion. Feeder cell batches are prepared by leukocyte removal from whole blood obtained from individual donors. The leukocyte removal product is centrifuged, washed, irradiated, and cryopreserved under GMP conditions on a Ficoll-Hypaque apparatus.

Importantly, live feeder cells should not be infused into patients receiving TIL therapy, as this can cause graft-versus-host disease (GVHD). Consequently, feeder cell growth is inhibited by gamma irradiation, leading to double-strand DNA breaks and loss of MNC cell viability upon reculture.

Feeder cell batches were evaluated according to two criteria: (1) their ability to amplify TILs by >100-fold in co-culture, and (2) their insufficient replication capacity.

Feeder cell batches were tested in a mini-REP format using two primary pre-REP TIL lines grown in vertical T25 tissue culture flasks. Feeder cell batches were tested against two distinct TIL lines, each with a unique ability to proliferate in response to activation within the REP. As a control, a number of previously demonstrated irradiated MNC feeder cells meeting the above criteria were processed with the test batches.

A stock solution of the same pre-REP TIL line is available to test all conditions and all feeder cell batches, ensuring that all batches tested in a single experiment undergo equivalent testing.

For each batch of feeder cells tested, a total of six T25 culture flasks were used: pre-REP TIL line #1 (2 flasks); pre-REP TIL line #2 (2 flasks); and feeder cell control (2 flasks). Flasks containing TIL lines #1 and #2 were used to evaluate the feeder cell batch's ability to amplify TILs. The feeder cell control flasks were used to evaluate insufficient replication capacity in the feeder cell batch.

A. Experimental Protocol

On day -2/3, thaw the TIL culture . Prepare CM2 medium and warm it in a 37°C water bath. Prepare 40 mL of CM2 supplemented with 3000 IU/mL IL-2. Keep warm until used. Place 20 mL of pre-warmed CM2 (without IL-2) into each of two 50 mL conical tubes labeled with the name of the TIL culture used. Remove the two designated pre-REP TIL cultures from the LN2 reservoir and transfer the vials to the tissue culture room. Thaw the vials by placing them in a sealed zip-lock storage bag in a 37°C water bath until a small amount of ice remains.

Using a sterile pipette, immediately transfer the contents of each vial to 20 mL of CM2 in a prepared, labeled 50 mL conical tube. Make up to 40 mL with IL-2-free CM2 to wash the cells and centrifuge at 400×CF for 5 minutes. Aspirate the supernatant and resuspend the cells in 5 mL of warm CM2 supplemented with 3000 IU/mL IL-2.

Take two aliquots (20 μL each) for cell counting using an automated cell counter. Record the counts. During counting, place a 50 mL conical tube containing TIL cells in a humidified 37°C, 5% _CO2 incubator with the cap loosened to allow gas exchange. Determine the cell concentration and dilute the TIL to 1 × ^10⁶ cells/mL in CM₂ supplemented with 3000 IU/mL IL-2.

In a humidified 37°C incubator, culture at 2 ml/well in multiple wells of a 24-well tissue culture plate as needed until day 0 of the micro-REP. Culture different TIL lines in separate 24-well tissue culture plates to avoid confusion and potential cross-contamination.

Day 0, start the micro-REP . Prepare sufficient CM2 medium for the number of feeder cell batches to be tested. (For example, prepare 800 mL of CM2 medium for testing 4 feeder cell batches at once). Divide a portion of the CM2 prepared above into equal parts and supplement it with 3000 IU/mL IL-2 for cell culture. (For example, prepare 500 mL of CM2 medium with 3000 IU/mL IL-2 for testing 4 feeder cell batches at once).

To prevent cross-contamination, the 24-well plates containing TIL cultures were removed from the incubator and transferred to the BSC.

Using sterile pipettes or 100-1000 μL pipettes and tips, remove approximately 1 mL of culture medium from each well to be used and place it into an unused well of a 24-well tissue culture plate.

Using fresh, sterile pipettes or 100-1000 μL pipettes and tips, mix the remaining culture medium with the TIL in the wells to resuspend the cells, and then transfer the cell suspension to 50 mL conical tubes labeled with the TIL batch name and record the volume.

Wash each well with the reserved culture medium and transfer the volume to the same 50 mL conical tube. Rotate the cells at 400 × CF to collect the cell pellet. Drain the culture supernatant and resuspend the cell pellet in 2–5 mL of CM2 medium containing 3000 IU/mL IL-2. The volume to be used is based on the number of wells collected and the size of the pellet; that is, the volume should be sufficient to ensure a concentration >1.3 × ^10⁶ cells/mL.

Using a serum pipette, thoroughly mix the cell suspension and record the volume. Take 200 μL for cell counting using an automated cell counter. During counting, place a 50 mL conical tube containing TIL cells in a humidified 5% _CO2 incubator at 37°C with the cap loosened to allow gas exchange. Record the count.

Remove the 50 mL conical tube containing TIL cells from the incubator and resuspend the cells at a concentration of 1.3 × ^10⁶ cells/mL in warm CM2 supplemented with 3000 IU/mL IL-2. Place the 50 mL conical tube back into the incubator and loosen the cap.

For the second TIL system, repeat the above steps.

Before plating TIL into the T25 culture flasks used for experiments, dilute TIL 1:10 to a final concentration of 1.3 × ^10⁵ cells/mL as shown below.

Prepare a working solution of pure CD3 (OKT3) from MACS GMP . Remove the OKT3 stock solution (1 mg/mL) from the 4°C freezer and place it in BSC. Use OKT3 at a final concentration of 30 ng/mL in the micro-REP medium.

In each T25 culture flask used in the experiment, 600 ng OKT3 is required per 20 mL; this is equivalent to 60 μL of 10 μg/mL solution per 20 mL, or 360 μL for all 6 culture flasks tested for each feeder cell batch.

For each feeder cell batch tested, prepare 400 μL of a 1:100 dilution of 1 mg/mL OKT3 for a working concentration of 10 μg/mL (e.g., for a single test of 4 feeder cell batches, prepare 1600 μL of a 1:100 dilution of 1 mg/mL OKT3: 16 μL of 1 mg/mL OKT3 + 1.584 mL of CM2 medium with 3000 IU/mL IL-2).

Prepare T25 culture flasks . Label each culture flask and fill it with CM2 medium before preparing feeder cells. Place the flasks in a humidified 5% _CO2 incubator at 37°C to keep the medium warm while awaiting the addition of the remaining components. After preparing feeder cells, add the components to the CM2 in each culture flask.

Other information is provided in Table 36.

Table 36. Solution Information.

Prepare feeder cells . For this protocol, each batch tested requires at least 78 × ^10⁶ feeder cells. Each 1 mL vial frozen by SDBB has 100 × ^10⁶ viable cells at freezing. Assuming a 50% recovery rate after thawing from a liquid _N₂ reservoir, it is recommended to thaw at least two 1 mL vials of feeder cells per batch, thus using an estimated 100 × ^10⁶ viable cells per REP. Alternatively, if supplied in 1.8 mL vials, a single vial may provide sufficient feeder cells.

Before thawing the feeder cells, pre-warm approximately 50 mL of IL-2-free CM2 for each feeder cell batch to be tested. Remove the designated feeder cell batch vial from the LN2 reservoir, place it in a zip-lock storage bag, and keep it on ice. Thaw the vial in the sealed zip-lock storage bag by immersion in a 37°C water bath. Remove the vial from the zip-lock bag, spray or wipe it with 70% EtOH, and transfer it to a BSC.

Using a pipette, immediately transfer the contents of the feeder cell vial to 30 mL of warm CM2 in a 50 mL conical tube. Wash the vial with a small volume of CM2 to remove any residual cells and centrifuge at 400×CF for 5 min. Aspirate the supernatant and resuspend the cells in 4 mL of warm CM2 with 3000 IU/mL IL-2. Take 200 μL for cell counting using an automated cell counter. Record the count.

Cells were resuspended at 1.3 × ^10⁷ cells/mL in warm CM2 with 3000 IU/mL IL-2. TIL cells were diluted from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL.

Set up the co-culture . Dilute TIL cells from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL. Add 4.5 mL of CM2 medium to a 15 mL conical tube. Remove the TIL cells from the incubator and resuspend them thoroughly using a 10 mL serum pipette. Take 0.5 mL of the 1.3 × ^10⁶ cells/mL TIL suspension and add it to 4.5 mL of medium in a 15 mL conical tube. Return the TIL stock solution vial to the incubator. Mix thoroughly. Repeat the above steps for the second TIL line.

Transfer the culture flasks containing pre-warmed medium for a single feeder cell batch from the incubator to the BSC. Mix the feeder cells by pipetting several times up and down with a 1 mL pipette tip, and transfer 1 mL (1.3 × ^10⁷ cells) to each culture flask containing the feeder cell batch. Add 60 μL of OKT3 working stock solution (10 μg/mL) to each culture flask. Return the two control flasks to the incubator.

Transfer 1 mL (1.3 × ^10⁵ ) of each TIL batch to a correspondingly labeled T25 culture flask. Place the flask back into the incubator and incubate upright. Begin the intervention on day 5. Repeat this procedure for all feeder cell batches tested.

On day 5, the culture medium was changed . CM2 with 3000 IU/mL IL-2 was prepared. 10 mL was needed for each culture flask. Using a 10 mL pipette, 10 mL of warm CM2 with 3000 IU/mL IL-2 was transferred to each culture flask. The culture flasks were returned to the incubator and incubated upright until day 7. The above procedure was repeated for all tested feeder cell batches.

Day 7, collect the culture . Remove the culture flasks from the incubator and transfer them to BSC, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove 10 mL of culture medium from each test flask and 15 mL of culture medium from each control flask.

Using a 10 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using the appropriate standard operating procedure with an automated cell counter. Record the counts on day 7. Repeat this procedure for all feeder cell batches tested.

The replication capacity of the feeder cell control flasks was insufficient, and the fold expansion of the culture flasks containing TILs was evaluated starting from day 0.

On day 7, continue processing the feeder cell control bottles until day 14. After counting the feeder cells on day 7, add 15 mL of fresh CM2 medium containing 3000 IU/mL IL-2 to each control bottle. Return the control bottles to the incubator and incubate them upright until day 14.

Day 14, extended non-proliferative phase of the feeder cell control flasks . Remove the culture flasks from the incubator and transfer them to BSCs, taking care to avoid disturbing the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove approximately 17 mL of culture medium from each control flask. Using a 5 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. Record the volume of each culture flask.

After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using an automated cell counter device following the appropriate standard operating procedure and record the count. Repeat this procedure for all feeder cell batches tested.

B. Results and Acceptance Criteria

Results . The dose of gamma irradiation was sufficient to prevent feeder cells from replicating. All batches were expected to meet the evaluation criteria and also showed a reduction in the total number of viable feeder cells remaining on day 7 of REP culture compared to day 0. All feeder cell batches were expected to meet the following evaluation criteria: 100-fold growth expansion of TILs up to day 7 of REP culture. The feeder cell control flask counts on day 14 were expected to continue the non-proliferative trend visible on day 7.

Acceptance Criteria . Each replicated TIL line for each batch of feeder cells must meet the following acceptance criteria. The acceptance criteria are doubled, as shown in Table 37 below.

Table 37. Implementation plan for acceptance criteria.

The study evaluated whether the irradiation dose was sufficient to prevent MNC feeder cells from replicating when cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. Insufficient replication capacity was assessed by total live cell count (TVC) as determined by automated cell counting at days 7 and 14 of the repetitive period (REP).

The acceptance criterion was "no growth," meaning that on days 7 and 14, the total number of viable cells did not increase compared to the initial number of viable cells placed in the culture on day 0 of the REP.

The ability of feeder cells to support TIL expansion was evaluated. TIL growth was measured by the fold increase in live cells from day 0 of the Reproductive Phase (REP) to day 7 of the REP. On day 7, the TIL culture reached a minimum 100-fold expansion (i.e., more than 100 times the total number of live TIL cells introduced into the culture on day 0 of the REP), as evaluated by automated cell counting.

Emergency testing of MNC feeder cell batches that do not meet acceptance criteria . If an MNC feeder cell batch fails to meet any of the acceptance criteria outlined above, the following steps will be performed to retest the batch to rule out simple experimenter error as the cause.

If two or more satellite testing vials remain in the batch, then the batch shall be tested again. If one satellite testing vial remains in the batch, or if no satellite testing vials remain in the batch, then the batch is deemed unqualified according to the acceptance criteria listed above.

For the materials to be considered合格 (qualified), both the relevant batch and the control batch must meet the above acceptance criteria. Only after meeting these criteria is the batch permitted for use.

Example 4: Preparation of IL-2 stock solution

This embodiment describes a process for dissolving purified lyophilized recombinant human interleukin-2 in a stock sample suitable for use in other tissue culture protocols (including all those described in this application and in the embodiments), including those involving the use of rhIL-2.

Procedure. Prepare a 0.2% acetic acid solution (HAc). Transfer 29 mL of sterile water to a 50 mL conical tube. Add 1 mL of 1N acetic acid to the 50 mL conical tube. Mix thoroughly by inverting the tube 2-3 times. Sterilize the HAc solution by filtration using a Steriflip filter.

Prepare PBS containing 1% HSA. Add 4 mL of 25% HSA stock solution to 96 mL of PBS in a 150 mL sterile filter unit. Filter the solution. Store at 4°C. Complete the form for each vial of rhIL-2 prepared.

Prepare a stock solution of rhIL-2 (final concentration of 6 × ^10⁶ IU/mL). Each batch of rhIL-2 is different, and the required information can be found in the manufacturer's Certificate of Analysis (COA), such as: 1) the mass of rhIL-2 per vial (mg), 2) the specific activity of rhIL-2 (IU/mg), and 3) the recommended 0.2% HAc reduction volume (mL).

Calculate the required volume of 1% HSA for a batch of rhIL-2 using the following formula:

For example, according to the COA of rhIL-2 batch 10200121 (Cellgenix), the specific activity of a 1 mg vial is 25 × ^10⁶ IU/mg. It is recommended to reconstitute rhIL-2 in 2 mL of 0.2% HAc.

Wipe the rubber stopper of the IL-2 vial with an alcohol swab. Using a 16G needle attached to a 3mL syringe, inject the recommended volume of 0.2% HAc into the vial. Be careful not to remove the stopper while withdrawing the needle. Invert the vial three times and rotate until all powder dissolves. Carefully remove the stopper and place it on the alcohol swab. Add the calculated volume of 1% HSA to the vial.

Store rhIL-2 solution. For short-term storage (<72 hours), store vials at 4°C. For long-term storage (>72 hours), aliquot the vials into smaller volumes and store them in frozen vials at -20°C until ready for use. Avoid freeze/thaw cycles. Expiry date is 6 months after preparation date. The Rh-IL-2 label includes the supplier and catalog number, lot number, expiry date, operator initials, concentration, and aliquot volume.

Example 5: Cryopreservation Process

This embodiment describes a cryopreservation process for TILs prepared according to the procedures described herein, using a CryoMed controlled-rate cryostat (Thermo Scientific) of model 7454.

The equipment used is as follows: aluminum box holder (compatible with CS750 freezer bags), freezer box for 750mL bags, low-pressure (22psi) liquid nitrogen tank, freezer, thermocouple sensor (for the strip type of bag) and CryoStore CS750 freezer bags (OriGen Scientific).

From nucleation to -20°C, the freezing process provides a rate of 0.5°C and a cooling rate of 1°C/min until the final temperature of -80°C. The program parameters are as follows: Step 1—Wait at 4°C; Step 2: 1.0°C/min (sample temperature) to -4°C; Step 3: 20.0°C/min (chamber temperature) to -45°C; Step 4: 10.0°C/min (chamber temperature) to -10.0°C; Step 5: 0.5°C/min (chamber temperature) to -20°C; and Step 6: 1.0°C/min (sample temperature) to -80°C.

Example 6: Tumor amplification process using a defined culture medium

The procedures disclosed above can be performed using appropriate culture media (e.g., CTS ^™ OpTmizer ^™ T-cell expansion SFM, ThermoFisher, including, for example, DM1 and DM2) instead of CM1 and CM2 media.

Example 7: Exemplary GEN 2 preparation of cryopreserved TIL cell therapy

This embodiment describes cGMP manufacturing of Iovance Biotherapeutics' TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices. This embodiment describes an exemplary cGMP manufacturing of a TIL cell therapy process in G-REX culture flasks according to current Good Tissue Practices and current Good Manufacturing Practices.

Table 38. Exemplary plans for process expansion.

Table 39. Culture Flask Volume

Day 0, CM1 medium preparation. In BSC, add reagents to RPMI 1640 culture bottles. Add the following reagents per bottle: heat-inactivated human AB serum (100.0 mL); GlutaMax ^™ (10.0 mL); gentamicin sulfate, 50 mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL).

Remove unnecessary materials from the BSC. Dispense culture medium reagents from the BSC, leaving gentamicin sulfate and HBSS in the BSC for use in preparing wash medium.

Thaw the IL-2 aliquot. Thaw a 1.1 mL IL-2 aliquot (6 × ^10⁶ IU/mL) (BR71424) until all ice has melted. Record the IL-2 batch number and expiration date.

Transfer the IL-2 stock solution to the culture medium. In BSC, transfer 1.0 mL of the IL-2 stock solution to the prepared CM1 Day 0 culture medium bottle. Add one bottle of CM1 Day 0 culture medium and 1.0 mL of IL-2 (6 × 10⁶ IU/mL).

Transfer G-REX100MCS to BSC. Aseptically transfer G-REX100MCS (W3013130) to BSC.

Pump all complete CM1 Day 0 culture medium into G-REX100MCS culture flasks. Alternatively, use conical tubes or G-Rex100MCS for tissue fragments.

Day 0, preparation of tumor washing medium. In BSC, 5.0 mL of gentamicin (W3009832 or W3012735) was added to a 1×500 mL HBSS medium (W3013128) bottle. Each bottle contained: HBSS (500.0 mL); gentamicin sulfate, 50 mg/mL (5.0 mL). Filtered HBSS containing gentamicin was prepared via a 1 L 0.22 μm filter unit (W1218810).

Day 0 Tumor Treatment. Obtain tumor samples and immediately transfer them to a kit at 2–8°C for treatment. Aliquot the tumor washing medium. Tumor washing 1 was performed using 8” forceps (W3009771). The tumor was removed from the sample vial and transferred to the prepared “Wash 1” culture dish. This was followed by tumor washing 2 and tumor washing 3. The tumor was measured and evaluated. It was assessed whether >30% of the total tumor area was observed to be necrotic and/or fatty tissue. Clean dissection was performed where applicable. If the tumor was large and >30% of the tissue surface was observed to be necrotic/fatty, “cleaning segmentation” was performed by removing the necrotic/fatty tissue using a combination of scalpel and/or forceps while preserving the internal structure of the tumor. The tumor was dissected. Using a combination of scalpel and/or forceps, the tumor sample was cut into an even number of appropriately sized fragments (up to 6 intermediate fragments). Intermediate tumor fragments were transferred. The tumor fragments were segmented into pieces approximately 3×3×3 mm in size. The intermediate fragments were stored to prevent dehydration. The intermediate fragment segmentation was repeated. The number of small pieces collected was determined. If only the desired tissue was preserved, additional favorable tumor fragments were selected from the “Favorable Intermediate Fragments” 6-well plate to fill the discarded fragments, so that up to 50 fragments were collected.

Prepare the conical tube. Transfer the tumor slice to a 50 mL conical tube. Prepare the BSC for G-REX100MCS. Remove the G-REX100MCS from the incubator. Aseptically transfer the G-REX100MCS culture flask to the BSC. Add the tumor fragment to the G-REX100MCS culture flask. Distribute the fragments evenly.

Incubate G-REX100MCS with the following parameters: G-REX culture flask: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Calculate: Incubation time; Lower limit = Incubation time + 252 hours; Upper limit = Incubation time + 276 hours.

After the process is complete, discard all remaining warm culture medium and thaw the aliquots of IL-2.

Day 11 - Culture medium preparation. Incubator monitoring. Incubator parameters: Temperature LED display: 37.0±2.0℃; CO2 percentage: 5.0±1.5% CO2.

Incubate 3 x 1000 mL RPMI 1640 medium (W3013112) bottles and 3 x 1000 mL AIM-V (W3009501) bottles in an incubator for ≥30 minutes. Remove the RPMI 1640 medium from the incubator. Prepare the RPMI 1640 medium. Filter the medium. Thaw 3 x 1.1 mL IL-2 aliquots (6 x 10⁶ IU/mL) (BR71424). Remove the AIM-V medium from the incubator. Add IL-2 to the AIM-V. Aseptically transfer the medium to the BSC using a 10 L Labtainer bag and relay pump transfer device.

Prepare a 10L Labtainer culture medium bag. Prepare a Baxa pump. Prepare another 10L Labtainer culture medium bag. Pump the culture medium into the 10L Labtainer. Remove the automatic pump tubing from the Labtainer bag.

Mix the culture medium. Gently knead the bag to mix. Sampling the culture medium according to the sampling plan. Take 20.0 mL of culture medium and place it in a 50 mL conical tube. Prepare the cell counting dilution tubes. In the BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" and lot number to four 15 mL conical tubes. Transfer the reagents from the BSC to 2–8°C. Prepare the 1 L transfer pack. On the outside of the BSC, fuse the 1 L transfer pack (according to process note 5.11) to the transfer device attached to the prepared "Complete CM2 Day 11 Medium" bag. Prepare the feeder cell transfer pack. Incubate the Complete CM2 Day 11 Medium.

Day 11 - TIL collection. Pretreatment form. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX100MCS from the incubator. Prepare 300mL transfer packs. Fuse the transfer packs to the G-REX100MCS.

Prepare the culture flask for TIL collection and begin the collection. Collect TILs. Using GatheRex, transfer the cell suspension through a blood filter to a 300 mL transfer pack. Examine the adherent cells on the membrane.

Rinse the culture flask membrane. Close the clamps on the G-REX100MCS. Ensure all clamps are closed. Heat-seal the TIL and "supernatant" transfer pack. Calculate the volume of the TIL suspension. Prepare the supernatant transfer pack for sampling.

Draw Bac-T samples. In the BSC, aspirate approximately 20.0 mL of supernatant from the 1 L “Supernatant” transfer pack and dispense into sterile 50 mL conical tubes.

Inoculate with BacT according to the sampling plan. Using an appropriately sized syringe, take 1.0 mL of sample from a prepared 50 mL conical tube labeled with BacT and inoculate it into an anaerobic bottle.

Incubate the TIL. Place the TIL transfer kit in an incubator only when needed. Perform cell counting and calculations. Measure the mean viable cell concentration and mean cell viability for each cell count. Viability ÷ 2. Viable cell concentration ÷ 2. Measure the upper and lower limits of the count. Lower limit: mean viable cell concentration × 0.9. Upper limit: mean viable cell concentration × 1.1. Confirm that both counts are within acceptable limits. Measure the mean viable cell concentration based on all four counts performed.

Adjusting the volume of the TIL suspension: Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume (A). Volume of the removed cell count sample (4.0 mL) (B) Adjusted total TIL cell volume C = A - B.

Calculate the total number of viable TIL cells. Average viable cell concentration*: total volume; total number of viable cells: C = A × B.

Flow cytometry calculation: If the total count of live TIL cells is ≥4.0× ^10⁷ , then calculate the volume of 1.0× ^10⁷ cells for the flow cytometry sample.

Total number of live cells required for flow cytometry: 1.0 × ^10⁷ cells. Cell volume required for flow cytometry: live cell concentration divided by 1.0 × ^10⁷ cells A.

Calculate the volume of a TIL suspension equal to 2.0 × ^10⁸ live cells. Calculate the volume of excess TIL cells to be removed as needed, and remove the excess TIL and place it in an incubator as needed. Calculate the total amount of excess TIL to be removed as needed.

The calculated volume of CS-10 medium added to the excess TIL cells at the target cell concentration for freezing is 1.0 × ^10⁸ cells/mL. Centrifuge the excess TIL as needed. Observe the conical tube and add CS-10.

Fill vials. Divide 1.0 mL of cell suspension into appropriately sized vials. Divide the remaining volume into appropriately sized vials. If the volume is ≤0.5 mL, add CS10 to the vials until the volume is 0.5 mL.

Calculate the cell volume required for 1 × ^10⁷ cells for cryopreservation. Remove the sample for cryopreservation. Place the TIL in an incubator.

Cryopreservation of samples. Observe the conical tube and slowly add CS-10, recording the volume of 0.5 mL of CS-10 added.

Day 11 - Feeder Cells. Obtain feeder cells. Obtain at least two bags of feeder cells from two different batches using an LN2 freezer. Preserve the cells on dry ice before thawing. Prepare a water bath or cryotherm. Thaw the feeder cells in a water bath or cryotherm at 37.0 ± 2.0 °C for approximately 3–5 minutes or until the ice has just melted. Remove the culture medium from the incubator. Combine the thawed feeder cells. Add the feeder cells to the transfer packs. Dispense the feeder cells from the syringe into the transfer packs. Mix the combined feeder cells and label the transfer packs.

Calculate the total volume of the feeder cell suspension in the transfer pack. Remove the cell counting samples. Using a separate 3 mL syringe, aspirate 4 × 1.0 mL of cell counting sample from the feeder cell suspension transfer pack using the non-essential injection port for each sample. Aliquot each sample into labeled cryovials. Perform cell counting, determine the multiplier, select a protocol, and enter the multiplier. Measure the mean viable cell concentration and mean cell viability for the cell count. Determine the upper and lower limits of count and confirm that they are within these limits.

Adjust the volume of the feeder cell suspension. Calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Calculate the total number of viable feeder cells. Obtain additional feeder cells as needed. Thaw additional feeder cells as needed. Place the fourth feeder cell bag in a zip-lock bag and thaw in a 37.0±2.0°C water bath or Cytotherm for approximately 3–5 minutes, and combine with the additional feeder cells. Measure the volume. Measure the volume of feeder cells in the syringe and record it below (B). Calculate the new total volume of feeder cells. Add the feeder cells to the transfer pack.

Prepare diluents as needed, adding 4.5 mL of AIM-V medium to four 15 mL conical tubes. Prepare for cell counting. Using a separate 3 mL syringe for each sample, remove 4 × 1.0 mL of the cell count sample from the feeder cell suspension transfer pack using the non-essential injection port. Perform cell counting and calculations. Determine the mean viable cell concentration based on all four counts performed. Adjust the feeder cell suspension volume and calculate the adjusted volume of the feeder cell suspension after removing the cell count sample. Subtract the removed 4.0 mL from the total feeder cell volume. Calculate the volume of feeder cell suspension required to obtain 5 x ^10⁹ viable feeder cells. Calculate the excess feeder cell volume. Calculate the volume of excess feeder cells to be removed. Remove the excess feeder cells.

Using a 1.0 mL syringe and a 16G needle, aspirate 0.15 mL of OKT3 and add OKT3. Heat-seal the feeder cell suspension transfer pack.

Day 11: G-REX Filling and Inoculation Setup for G-REX500MCS. Remove the "Complete CM2 Day 11 Medium" from the incubator and pump the medium into the G-REX500MCS. Pump 4.5L of medium into the G-REX500MCS, filling to the marked line on the culture flask. Heat seal as needed and incubate the culture flask. Fuse the feeder cell suspension transfer kit to the G-REX500MCS. Add feeder cells to the G-REX500MCS. Heat seal. Fuse the TIL suspension transfer kit to the culture flask. Add TIL to the G-REX500MCS. Heat seal. Incubate the G-REX500MCS at 37.0±2.0℃, CO2 percentage: 5.0±1.5% CO2.

Calculate the incubation window. Perform calculations to determine the appropriate time to remove the G-REX500MCS from the incubator on day 16. Lower limit: Incubation time + 108 hours. Upper limit: Incubation time + 132 hours.

Day 11: Cryopreservation of Excess TIL. Applicable to: Cryopreservation of excess TIL vials. Confirm that the Controlled Rate Freezer (CRF) has been set before freezing. Perform cryopreservation. Transfer the vial from the controlled rate freezer to the appropriate storage container. After freezing is complete, transfer the vial from the CRF to the appropriate storage container. Transfer the vial to the appropriate storage container. Record the storage location in LN2.

Day 16 Culture Medium Preparation. Preheat AIM-V medium. Calculate the warming time for culture bags 1, 2, and 3. Ensure all bags have been warmed for 12 to 24 hours. Set up the 10L Labtainer for supernatant. Attach the larger diameter end of the fluid pump transfer device to a concave port of the 10L Labtainer bag using a Luer connector. Set up and label the 10L Labtainer for supernatant. Set up the 10L Labtainer for supernatant. Ensure all clamps are closed before removing from the BSC. Note: Use the supernatant bag during TIL collection, which can be performed in parallel with culture medium preparation.

Thaw IL-2. Thaw 5 × 1.1 mL IL-2 aliquots (6 × ^10⁶ IU/mL) (BR71424) of each CTS AIM V medium bag until all ice melts. Aliquot 100.0 mL GlutaMax ^™ . Add IL-2 to GlutaMax ^™ . Prepare the CTS AIM V medium bag for preparation. Prepare the CTS AIM V medium bag for preparation. Multistage Baxa pump. Prepare the medium. Pump GlutaMax ^™ + IL-2 into the bag. Monitor parameters: Temperature LED display: 37.0 ± 2.0 °C, _CO₂ percentage: 5.0 ± 1.5% _CO₂ . Warm up the complete CM4 day 16 medium. Prepare the dilution.

Day 16, REP splitting. Incubator parameters monitored: Temperature LED display: 37.0±2.0℃, _CO2 percentage: 5.0±1.5% _CO2 . Remove G-REX500MCS from the incubator. Prepare a 1L transfer pack, label it TIL suspension, and weigh it as 1L.

The volume of the G-REX500MCS decreased. Approximately 4.5 L of culture supernatant was transferred from the G-REX500MCS to a 10 L Labtainer.

Prepare the culture flask for TIL collection. After removing the supernatant, close all clamps leading to the red tubing.

Initiate TIL collection . Vigorously tap the culture flask and rotate the culture medium to detach the cells, ensuring all cells are detached.

TIL Collection . Loosen all clamps leading to the TIL suspension transfer pack. Using GatheREX, transfer the cell suspension into the TIL suspension transfer pack. Note: Ensure the edges are kept tilted until all cells and culture medium are collected. Check for adherent cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS. Heat-seal the transfer pack containing TIL. Heat-seal the 10L Labtainer containing the supernatant. Record the weight of the transfer pack containing the cell suspension and calculate the suspension volume. Prepare the transfer pack for sample removal. Remove the test sample from the cell supernatant.

Aseptic and BacT test sampling . Take 1.0 mL of sample from the prepared 15 mL cone-labeled BacT. Take the cell counting sample. In the BSC, use a separate 3 mL syringe to take 4 × 1.0 mL cell counting samples from the TIL suspension transfer pack for each sample.

Remove the Mycoplasma sample. Using a 3 mL syringe, remove 1.0 mL from the TIL suspension transfer pack and place it into a prepared 15 mL conical tube labeled “Mycoplasma diluent”.

Prepare the transfer kit for inoculation. Place the TIL in an incubator. Remove the cell suspension from the BSC and place it in an incubator until needed. Perform cell counting and calculation. First, dilute the cell count sample to a 1:10 dilution by adding 0.5 mL of the cell suspension to 4.5 mL of prepared AIM-V medium. Measure the mean viable cell concentration and mean survival rate of the cells used for cell counting. Measure the upper and lower limits of counting. Note: Dilutions can be adjusted based on the expected cell concentration. Determine the mean viable cell concentration from all four counts performed. Adjust the volume of the TIL suspension. Calculate the adjusted volume of the TIL suspension after removing the cell count sample. Total TIL cell volume minus the 5.0 mL removed for testing.

Calculate the total number of viable TIL cells. Calculate the total number of culture flasks to be inoculated. Note: The maximum number of G-REX500MCS culture flasks to be inoculated is five. If the calculated number of culture flasks to be inoculated exceeds five, inoculate only five culture flasks using all available volumes of cell suspension.

Calculate the number of culture flasks needed for subculture. Calculate the number of culture medium bags still needed besides the prepared bags. Prepare one 10L "CM4 Day 16 Medium" bag for every two G-REX-500M culture flasks as calculated. Continue inoculating the first G-REX-500M culture flask while preparing additional medium and warming it. Prepare the determined number of additional culture medium bags and warm them. Fill the G-REX500MCS. Prepare the pumpable medium and pump 4.5L of medium into the G-REX500MCS. Heat seal. Repeat filling. Incubate the culture flasks. Calculate the target volume of TIL suspension to be added to the new G-REX500MCS culture flasks. If the calculated number of culture flasks exceeds five, inoculate only five culture flasks using all volumes of cell suspension. Prepare the culture flasks for inoculation. Remove the G-REX500MCS from the incubator. Prepare the G-REX500MCS for pumping. Close all clamps except for the larger filter line. Remove the TIL from the incubator. Prepare the cell suspension for inoculation. Aseptically fuse the "TIL suspension" transfer package (according to process note 5.11) to the pump inlet line. Place the TIL suspension bag on the scale.

Inoculate the culture flasks with TIL suspension. Pump the calculated volume of TIL suspension into the culture flasks. Heat seal. Fill the remaining culture flasks.

Monitoring the incubator. Incubator parameters: Temperature LED display: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Incubation culture bottles.

The time range for removing G-REX500MCS from the incubator on day 22 was determined.

Day 22 Wash Buffer Preparation. Prepare a 10L Labtainer bag. Attach a 4” plasma transfer device to the 10L Labtainer bag via a Luer connector in the BSC. Prepare the 10L Labtainer bag. Close all clamps before transferring out of the BSC. Note: Prepare one 10L Labtainer bag for every two G-REX 500MCS culture flasks to be collected. Aspirate the Plasmalyte pump into the 3000mL bag and remove air from the 3000mL Origen bag by inverting the pump and manipulating the bag's position. Add 25% human albumin to the 3000mL bag. Obtain a final volume of 120.0mL of 25% human albumin.

Prepare the IL-2 diluent. Using a 10 mL syringe, remove 5.0 mL of LOVO wash buffer through the needle-free injection port on the LOVO wash buffer bag. Dispense the LOVO wash buffer into 50 mL conical tubes.

Distribute LOVO wash buffer into equal portions of the CRF blank bags. Using a 100mL syringe, draw 70.0mL of LOVO wash buffer from the needleless injection port.

Thaw one 1.1 mL IL-2 (6 × 10⁶ IU/mL) until all ice has melted. Add 50 μL of IL-2 stock solution (6 × ^10⁶ IU/mL) to a 50 mL conical tube labeled “IL-2 Diluent”.

Preparation for cryopreservation . Place 5 cryopreservation boxes at 2-8°C to pretreat them for use in the final product cryopreservation.

Prepare the cell counting dilution. In a BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" to four separate 15 mL conical tubes. Prepare for cell counting. Label the four frozen vials with vial numbers (1-4). Store the vials in the BSC for later use.

TIL collection on day 22. Monitor the incubator. Incubator parameters: Temperature LED display: 37±2.0℃, CO2 percentage: 5%±1.5%. Remove the G-REX500MCS culture flask from the incubator. Prepare and label the TIL collection bag. Seal any additional connectors. Volume reduction: Transfer approximately 4.5L of supernatant from the G-REX500MCS to the supernatant bag.

Prepare the culture flask for TIL collection. Initiate TIL collection. Vigorously tap the culture flask and rotate the culture medium to detach the cells. Ensure all cells have detached. Initiate TIL collection. Loosen all clamps leading to the TIL suspension collection bag. TIL collection. Using GatheRex, transfer the TIL suspension to a 3000 mL collection bag. Examine the adhered cells on the membrane. Rinse the culture flask membrane. Close the clamps on the G-REX500MCS, ensuring all clamps are closed. Transfer the cell suspension to the LOVO source bag. Close all clamps. Heat seal. Remove 4 × 1.0 mL cell counting samples.

Perform cell counting. Cell counting and calculation were performed using NC-200 and process note 5.14. The cell counting sample was first diluted by adding 0.5 mL of cell suspension to 4.5 mL of prepared AIM-V medium, resulting in a 1:10 dilution. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The upper and lower limits of counting were determined. The mean viability, viable cell concentration, and total nucleated cell concentration of the cells used for counting were determined. The LOVO source bag was weighed. The total number of viable TIL cells was calculated. The total number of nucleated cells was calculated.

Prepare the Mycoplasma diluent. Take 10.0 mL from a supernatant bag via the Luhr sample port and place it into a 15 mL conical tube.

Perform the "TIL G-REX collection" protocol and determine the target volume of the final product. Load the disposable kit. Remove the filtrate bag. Enter the filtrate volume. Place the filtrate container on the lab bench. Attach PlasmaLyte. Confirm that PlasmaLyte has been attached and that it is moving. Attach the source container to the tubing and confirm that it has been attached. Confirm that PlasmaLyte is moving.

Final preparation and filling. Target volume/bag calculation. Calculate the volume of CS-10 and LOVO wash buffer to be prepared in blank bags. Prepare CRF blank bags.

Calculate the volume of IL-2 to be added to the final product. Required final IL-2 concentration (IU/mL) - 300 IU/mL. IL-2 working stock solution: 6 × ^10⁴ IU/mL. Assemble and connect the device. Aseptically fuse the 4S-4M60 to the CC2 unit connector. Aseptically fuse the CS750 cryoprotectant bag to the prepared wire bundle. Fuse the CS-10 bag to the tip of the 4S-4M60. Prepare TILs with IL-2. Using an appropriately sized syringe, extract the measured amount of IL-2 from the “IL-2 6 × ^10⁴ ” aliquot. Label the prepared TIL bag. Add the prepared TIL bag to the device. Add CS10. Switch syringes. Aspirate approximately 10 mL of air into a 100 mL syringe and replace the 60 mL syringe on the device. Add CS10. Prepare the CS-750 bag. Dispense cells.

Remove air from the final product bag and obtain the retention. Once the last final product bag has been filled, close all clamps. Draw 10 mL of air into a new 100 mL syringe and replace the syringe on the device. Dispense the retention into 50 mL conical tubes and label the tubes “Retention” and the batch number. Repeat the air removal steps for each bag.

Prepare the final product for cryopreservation, including visual inspection. Store the cryopreservation bag on a cooling pack or at 2-8°C before cryopreservation.

Remove the cell counting sample. Using an appropriately sized pipette, remove 2.0 mL of the retained sample and place it in a 15 mL conical tube for cell counting. Perform cell counting and calculation. Note: Only dilute one sample to an appropriate confirmatory dilution. Dilute the remaining sample to the appropriate dilution factor and continue counting. Measure the mean viable cell concentration and mean viability of the cells used for cell counting. Measure the upper and lower limits of count. Note: The dilution may be adjusted according to the expected cell concentration. Measure the mean viable cell concentration and mean viability. Measure the upper and lower limits of count. Calculate IFN-γ. Heat-seal the final product bag.

The following exemplary sampling plan is used to label and collect samples.

Table 40. Sample Plan.

Sterility and BacT test . Test sampling. In the BSC, take 1.0 mL of sample from the retained cell suspension collected using an appropriately sized syringe and inoculate it into an anaerobic flask. Repeat the above steps for the aerobic flask.

Cryopreservation of the final product. Prepare the controlled-rate freezer (CRF). Verify that the CRF is set up. Set the CRF probe. Place the final product and sample in the CRF. Measure the time required to reach 4℃ ± 1.5℃ and continue the CRF operation. Complete the CRF and store. Stop the CRF after completion. Remove the box and vial from the CRF. Transfer the box and vial to gas chromatography-tandem nitrogen (LN2) for storage. Record the storage location.

The final processing and analysis of the drug included the following tests: (Day 22) CD3+ cell count of REP on Day 22 by flow cytometry; (Day 22) Gram staining method (GMP); (Day 22) bacterial endotoxin test by gel clot LAL assay (GMP); (Day 16) BacT sterility assay (GMP); (Day 16) TD-PCR (GMP) detection of Mycoplasma DNA; acceptable appearance characteristics; (Day 22) BacT sterility assay (GMP); (Day 22) IFN-γ assay. Other potency assays as described herein were also used to analyze the TIL product.

Example 8: Exemplary Implementation of the GEN 3 Amplification Platform

Day 0

Prepare tumor washing medium. Warm the medium before starting. Add 5 mL of gentamicin (50 mg/mL) to a 500 mL HBSS bottle. Add 5 mL of tumor washing medium to a 15 mL Erlenmeyer flask for OKT3 dilution. Prepare feeder cell bags. Aseptically transfer feeder cells to feeder cell bags and store at 37°C until use or freeze. If stored at 37°C, count the feeder cells. If frozen, thaw and then count the feeder cells.

The optimal range for feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with a 4.5 mL LAIM-V. Add 0.5 mL of cell fraction for each cell count. If the total number of viable feeder cells is ≥ 1 × ^10⁹ cells, adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag to add 1 × ^10⁹ cells to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of tumor washing medium to an OKT3 aliquot (100 μL). Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the second feeder cell bag. Adjust the medium volume to a total volume of 2 L. Transfer the second feeder cell bag to an incubator.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 0: 15 μg/vial, i.e., 30 ng/mL in 500 mL, up to approximately 60 μL, 1 aliquot.

Add 5 mL of tumor wash medium to all wells of a 6-well plate labeled as excess tumor flakes. Reserve the tumor wash medium for further use in maintaining tumor hydration during dissection. Add 50 mL of tumor wash medium to each 100 mm Petri dish.

Using a ruler as a reference, divide the tumor into ^27mm segments (3×3×3mm) under the dissecting culture dish lid. Dissect the intermediate segments until 60 segments are obtained. Count the total number of final segments and prepare G-REX-100MCS culture flasks (usually 60 segments/flask) based on the number of final segments produced.

Preserve favorable tissue fragments in conical tubes labeled Fragment Tubes 1 through 4. Calculate the number of G-REX-100MCS culture flasks seeded with feeder cell suspension based on the number of initial fragment tubes.

Remove the feeder cell bag from the incubator and inoculate with G-REX-100 MCS. Label it D0 (day 0).

Add the tumor fragment to the culture in the G-REX-100 MCS . Under aseptic conditions, unscrew the caps of the G-REX-100 MCS labeled "Tumor Fragment Culture (D0) 1" and the 50 mL conical tube labeled "Fragment Tube". Rotate the opened fragment tube 1 while simultaneously slightly lifting the cap of the G-REX-100 MCS. Add the culture medium and fragment together to the G-REX-100 MCS while rotating. Record the number of fragments transferred to the G-REX-100 MCS.

Once the fragment is at the bottom of the GREX culture flask, aspirate 7 mL of culture medium and create seven 1 mL aliquots: 5 mL for extended characterization and 2 mL for sterility. Store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until needed.

Inoculate one anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle with 1 mL of the final fragment culture supernatant. Repeat the above procedure for each culture bottle from which samples are taken.

On the 7th-8th day

Prepare the feeder cell bags. If frozen, thaw the feeder cell bags in a 37°C water bath for 3–5 minutes. If frozen, count the feeder cells. The optimal feeder cell concentration is between 5 × ^10⁴ and 5 × ^10⁶ cells/mL. Prepare four conical tubes with 4.5 mL AIM-V. For each cell count, add 0.5 mL of the cell fraction to a new frozen tube. Mix the sample thoroughly and perform cell counting.

If the total number of live feeder cells is ≥2× ^10⁹ cells, proceed to the next step to adjust the feeder cell concentration. Calculate the volume of feeder cells removed from the first feeder cell bag so that 2× ^10⁹ cells can be added to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 μL of HBSS to 100 μL of OKT3 aliquot. Mix by pipetting three times, upwards and downwards. Prepare two aliquots.

OKT3 Formulation Details: OKT3 can be aliquoted and frozen in 100 μL aliquots from the original stock solution concentration (1 mg/mL) in the vial. Each 1 mL vial contains approximately 10 aliquots. Store at -80°C. Day 7/8: 30 μg/vial, i.e., 60 ng/mL in 500 mL, up to 120 μL, 2 aliquots.

Using a syringe and aseptic technique, draw 0.6 mL of OKT3 and add it to the feeder cell bag, ensuring all is added. Adjust the culture medium volume to a total of 2 L. Repeat the above steps for the second OKT3 aliquot and add it to the feeder cell bag. Transfer the second feeder cell bag to an incubator.

Prepare G-REX100MCS culture flasks containing feeder cell suspension . Record the number of G-REX100MCS culture flasks to be processed based on the number of G-REX culture flasks produced on day 0. Remove the G-REX culture flasks from the incubator and remove the second feeder cell bag from the incubator.

Remove the supernatant before adding the feeder cell suspension . Connect a 10 mL syringe to a G-REX100 culture flask and aspirate 5 mL of culture medium. Prepare five 1 mL aliquots, 5 mL for extended characterization, and store the five aliquots used for extended characterization (final fragment culture supernatant) at -20°C until requested by the initiator. Label each G-REX100 culture flask and repeat the above steps.

Depending on the number of culture flasks, 5-20 × 1 mL samples are used for characterization:

●5mL = 1 culture flask

●10mL = 2 culture flasks

●15mL = 3 culture flasks

●20mL = 4 culture flasks

Continue seeding feeder cells into G-REX100 MCS flasks, and repeat the above procedure for each G-REX100 MCS flask. Using aseptic transfer, transfer 500 mL of the second feeder cell bag by weight (assuming 1 g = 1 mL) to each G-REX-100 MCS flask and record the transfer volume. Label this as Day 7 culture and repeat the above procedure for each G-REX100 flask. Transfer the G-REX-100 MCS flasks to an incubator.

Days 10-11

Remove the first G-REX-100MCS culture flask and, under aseptic conditions, use a 10mL syringe to extract 7mL of pretreated culture supernatant. Prepare seven 1mL aliquots: 5mL for extended characterization and 2mL for sterile samples.

Carefully mix the culture flasks and use a new 10mL syringe to remove 10mL of the supernatant and transfer it to a 15mL tube labeled D10/11 Mycoplasma supernatant.

Carefully mix the culture flasks and, using a new syringe, dispense the following volumes based on the number of culture flasks to be processed:

● 1 bottle = 40mL

●2 bottles = 20mL/bottle

● 3 bottles = 13.3 mL/bottle

● 4 bottles = 10mL/bottle

Aspirate a total of 40 mL from all culture flasks and collect the samples in a 50 mL conical tube labeled ‘Day 10/11 QC Sample’. Store the tube in an incubator until needed. Perform cell counting and cell distribution.

Five aliquots (pretreated culture supernatant) used for extended characterization were stored at ≤-20°C until needed. One anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle were inoculated with 1 mL of pretreated culture supernatant, respectively.

Continue transferring the cell suspension to G-REX-500MCS and repeat the above procedure for each G-REX-100MCS. Under aseptic conditions, transfer the contents of each G-REX-100MCS to a G-REX-500MCS, monitoring approximately 100 mL of fluid transfer each time. Stop transferring when the volume of the G-REX-100MCS decreases to 500 mL.

During the transfer step, use a 10 mL syringe and aspirate 10 mL of cell suspension from the G-REX-100MCS into the syringe. Follow the instructions according to the number of flasks in culture. If only 1 flask: use two syringes to aspirate a total of 20 mL. If 2 flasks: aspirate 10 mL from each flask. If 3 flasks: aspirate 7 mL from each flask. If 4 flasks: aspirate 5 mL from each flask. Transfer the cell suspension to a common 50 mL conical tube. Keep in the incubator until the cell counting step and QC sample. The total number of cells required for QC is approximately 20e6 cells: 4 × 0.5 mL cell counts (cell counts are undiluted first).

The required cell quantity for the assay is as follows:

1. At least 10 × ^10⁶ cells should be used for efficacy assays, such as those described herein, or for IFN-γ or granzyme B assays.

2.1 × ^10⁶ cells were used for Mycoplasma.

3.5 × ^10⁶ cells were used for CD3+/CD45+ flow cytometry.

Transfer the G-REX-500MCS culture flask to an incubator.

Prepare QC samples . In this embodiment, at least 15 × ^10⁸ cells are required for the assay. Assays include: cell count and viability; Mycoplasma (1 × ^10⁶ cells/mean viable concentration); flow cytometry (5 × ^10⁶ cells/mean viable concentration); and IFN-γ assay (5 × ^10⁶ cells - 1 × ^10⁶ cells; IFN-γ assay requires 8-10 × ^10⁶ cells).

Calculate the volume of the cell fraction cryopreserved at 10 × ^10⁶ cells/mL, and calculate the number of vials required.

Days 16-17

Wash buffer preparation (1% HSA Plasmalyte A). Transfer the HSA and Plasmalyte to a 5L bag to prepare the LOVO wash buffer. Under aseptic conditions, transfer a total volume of 125 mL of 25% HSA to a 5L bag. Remove 10 mL or 40 mL of the wash buffer and transfer it to an 'IL-2 6× ^10⁴ IU/mL tube' (10 mL if the IL-2 is pre-prepared, or 40 mL if the IL-2 is freshly prepared).

Calculate the volume of restored IL-2 added to Plasmalyte + 1% HSA: Volume of restored IL-2 = (Final concentration of IL-2 × Final volume) / Specific activity of IL-2 (based on standard determination). The final concentration of IL-2 is 6 × ^10⁴ IU/mL. The final volume is 40 mL.

Take the calculated initial volume of IL-2 required for IL-2 recovery and transfer it to a tube labeled 'IL-2 6 × ^10⁴ IU/mL'. Add 100 μL of IL-2 6 × ^10⁶ IU/mL from the pre-prepared aliquot to a tube labeled 'IL-2 6 × ^10⁴ IU/mL' containing 10 mL of LOVO wash buffer.

Remove approximately 4500 mL of supernatant from the G-REX-500MCS culture flask. Rotate the remaining supernatant and transfer the cells to the cell collection pocket. Repeat the above steps for all G-REX-500MCS culture flasks.

Take 60 mL of the supernatant and add it to the supernatant tube for quality control determination, including mycoplasma detection. Store at +2–8°C.

Cell collection . Cell counting. Prepare four 15 mL conical tubes containing 4.5 mL of AIM-V. These conical tubes can be prepared in advance. Optimal range = between 5 × ^10⁴ and 5 × ^10⁶ cells/mL (1:10 dilution recommended). For a 1:10 dilution, add 500 μL of CF to the previously prepared 4500 μL of AIM-V. Record the dilution factor.

Calculate the total cells (TC) of pre-LOVO (survival + death) =

Average total cells

Concentration (pre-LOVO TC concentration)

(Survival + Death)

X

Volume of source bag

Calculate the TVC (total viable cells) of pre-LOVO (survival) =

Average total viable cells

Concentration (pre-LOVO TVC)

(Survival)

X

LOVO source bag volume

When the total cell count (TC) is >5 × ^10⁹ , remove 5 × ^10⁸ cells for cryopreservation as a reserve sample for MDA. 5 × ^10⁸ ÷ average TC concentration (step 14.44) = volume to be removed.

When the total cell count (TC) is ≤5× ^10⁹ , 4× ^10⁶ cells are removed and cryopreserved as a reserve sample for MDA. 4× ^10⁶ ÷ average TC concentration = volume to be removed.

When determining the total cell count, the number of cells to be removed should allow for the retention of 150 × ^10⁹ viable cells. Confirm the pre-LOVO TVC as 5 × ^10⁸ or 4 × ^10⁶ , or not applicable. Calculate the volume of cells to be removed.

Calculate the remaining total cells in the bag. Calculate the pre-LOVO TC (total cells). [Average total cell concentration x remaining volume = pre-LOVO remaining TC]

Select the corresponding process from Table 41 based on the total number of remaining cells.

Table 41. Total number of cells.

Select the volume of IL-2 to be added corresponding to the procedure used. The volume is calculated as follows: Residue volume × 2 × 300 IU/mL = Required IU of IL-2. Required IU of IL-2 / 6 × ^10⁴ IU/mL = Volume of IL-2 added after LOVO bagging. Record all added volumes. Obtain samples in frozen vials for further analysis.

Mix the cell products thoroughly. Seal all bags for further processing, including freezing where appropriate.

Perform endotoxin, IFN-γ, sterility, and other assays on the obtained frozen vial samples as needed.

Example 9: Exemplary Processes for GEN 2 and GEN 3

This example illustrates the Gen 2 and Gen 3 processes. The Gen 2 and Gen 3 TILs typically consist of autologous TILs derived from individual patients (via surgical tumor resection) and then expanded ex vivo. The first expansion step in initiating the Gen 3 process involves cell culture in the presence of interleukin-2 (IL-2) and the monoclonal antibody OKT3, which targets the T-cell co-receptor CD3 on the cytoskeleton of irradiated peripheral blood mononuclear cells (PBMCs).

The fabrication of the Gen 2 TIL product consists of two stages: 1) pre-rapid expansion (pre-REP) and 2) rapid expansion protocol (REP). During pre-REP, excised tumors are cut into ≤50 fragments of 2-3 mm in each dimension. These fragments are cultured for 11 days in serum-containing medium (RPMI 1640 medium supplemented with 10% HuSAB) and 6,000 IU/mL interleukin-2 (IL-2). On day 11, TILs are collected and introduced into a large-scale secondary REP expansion. The REP consists of the following: activation of ≤200 × ^10⁶ live cells from pre-REP for 5 days in ⁵ L of CM2 supplemented with 3,000 IU/mL rhIL-2, with the feeder cells loaded with 150 μg of monoclonal anti-CD3 antibody (OKT3). On day 16, the culture volume was reduced by 90%, and the cells were fractionated to ≥1× ^10⁹ viable lymphocytes/flask into multiple G-REX-500 culture flasks, and brought to a final volume of 5L with CM4. The TIL was then incubated for another 6 days. On day 22, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

The fabrication of Gen 3 TIL products consists of three stages: 1) initiation of the first expansion protocol, 2) rapid second expansion protocol (also known as the rapid expansion phase or REP), and 3) dissociation of subcultures. To achieve initiation of the first expansion TIL proliferation, the excised tumor was fragmented into ≤120 fragments, each 2-3 mm in dimensions. On day 0 of initiation of the first expansion, a feeder layer of approximately 2.5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was established on a surface area of approximately 100 ^cm² in each of three 100 MCS dishes. The tumor fragments were distributed in the three 100 MCS dishes and cultured for 7 days in CM1 medium containing 500 mL of serum and 6,000 IU/mL of interleukin-2 (IL-2) and 15 μg of OKT-3. On day 7, REP was initiated as follows: an additional feeder cell layer of approximately 5 × ^10⁸ allogeneic irradiated PBMC feeder cells loaded with OKT-3 was incorporated into the tumor fragmentation culture phase in each of three 100 MCS dishes and cultured with 500 mL CM2 medium and 6,000 IU/mL IL-2 and 30 μg OKT-3. REP initiation was enhanced by activating the entire initiation first expansion culture in the same dish, which was achieved by transferring OKT3-loaded feeder cells to the 100 MCS dishes using a closed-system fluid transfer. For Gen 3, TIL scaling up or splitting involved the following procedural steps: the entire cell culture was scaled up and transferred to the larger dish via a closed-system fluid transfer (from a 100 M culture flask to a 500 M culture flask) and an additional 4 L CM4 medium was added. On day 16, REP was collected, washed, prepared, and frozen, and then transported to the clinical site at -150°C for infusion.

Overall, the Gen 3 process is a shorter, more scalable, and easier-to-modify amplification platform that will be suitable for stable manufacturing and process comparability.

Table 50. Comparison of the manufacturing processes of Exemplary Gen 2 and Exemplary Gen 3.

On day 0, for both processes, tumor cells were washed three times and fragments were randomly grouped into two pools; one pool for each process. For the Gen 2 process, fragments were transferred to a GREX 100MCS flask containing 1 LCM1 of medium with 6,000 IU/mL rhIL-2. For the Gen 3 process, fragments were transferred to a G-REX100MCS culture flask containing 500 mL CM1 of medium with 6,000 IU/mL rhIL-2, 15 μg OKT-3, and 2.5 × ^10⁸ feeder cells. TILs were inoculated on Rep start day according to different days for each process. For the Gen 2 process, where the volume of the G-REX-100MCS culture flasks decreased by 90%, the collected cell suspension was transferred to new G-REX-500MCS on day 11 to initiate REP in CM2 medium containing IL-2 (3000 IU/mL) plus 5 × ^10⁹ feeder cells and OKT-3 (30 ng/mL). Cells were expanded according to the protocol and split into multiple G-REX-500MCS culture flasks containing CM4 medium and IL-2 (3000 IU/mL) on day 16. Cultures were then collected and cryopreserved on day 22 according to the protocol. For the Gen 3 process, REP initiation was performed on day 7 using the same G-REX-100MCS. In short, 500 mL of CM2 medium containing IL-2 (6000 IU/mL), 5 × ^10⁸ feeder cells, and 30 μg OKT-3 was added to each culture flask. On days 9–11, scale up the culture. Transfer the entire volume of G-REX100M (1 L) to G-REX-500MCS and add 4 L of CM4 containing IL-2 (3000 IU/mL). Incubate the flask for 5 days. On day 16, collect the culture and freeze it.

The comparison included three different tumors: two lung tumors (L4054 and L4055) and one melanoma tumor (M1085T).

For L4054 and L4055, CM1 (medium 1), CM2 (medium 2), and CM4 (medium 4) media were prepared in advance and maintained at 4°C. CM1 and CM2 media were prepared without filtration to compare cell growth with and without filtration of the media.

For L4055 tumors, the culture medium was heated at 37°C for up to 24 hours during REP initiation and scaling up.

Results . For the total number of viable cells achieved, the results for Gen 3 were within 30% of those for Gen 2. Upon restimulation, the Gen 3 final product exhibited higher IFN-γ production. As measured by the total number of unique CDR3 sequences present, the Gen 3 final product showed increased clonal diversity. The Gen 3 final product also exhibited a longer mean telomere length.

Pre-REP and REP expansion in Gen 2 and Gen 3 processes followed the procedures described above. For each tumor, both pools contained an equal number of fragments. Due to the smaller size of the tumors, the maximum number of fragments per flask could not be reached. Total pre-REP cells (TVCs) were collected and counted on day 11 of the Gen 2 process and day 7 of the Gen 3 process. To compare the two pre-REP groups, the cell count was divided by the number of fragments provided in the culture to calculate the average number of viable cells per fragment. As indicated in Table 51 below, more cells were consistently grown per fragment in the Gen 2 process compared to the Gen 3 process. The extrapolation of the expected TVC number on day 11 of the Gen 3 process was calculated by dividing the pre-REP TVC by 7 and then multiplying by 11.

Table 51. Pre-REP cell count

*L4055, unfiltered culture medium.

For both Gen 2 and Gen 3 processes, TVCs were counted according to process conditions, and the percentage of viable cells per day of the process was generated. At harvest, cells were collected on day 22 (Gen 2) and day 16 (Gen 3), and TVC counts were generated. The TVCs were then divided by the number of fragments provided on day 0 to calculate the average number of viable cells per fragment. The fold increase was calculated by dividing the collected TVCs by the REP initiation TVCs. As shown in Table 52, comparing Gen 2 and Gen 3, the fold increases were similar for L4054; in the case of L4055, the fold increase was higher for the Gen 2 process. Specifically, in this case, the medium was heated to 24°C prior to the REP initiation day. A higher fold increase was also observed in Gen 3 for M1085T. The extrapolation of the expected TVC count on day 22 of the Gen 3 process was calculated by dividing the REP TVCs by 16 and then multiplying by 22.

Table 52. Total viable cell count and fold expansion of TIL final products.

*L4055, unfiltered culture medium.

Table 53: Survival percentage of final TIL products: Final TIL REP products were compared to the survival percentage release criteria after collection. All conditions for Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Following collection, the final TIL REP product was compared against release criteria based on survival percentage. All conditions for both Gen 2 and Gen 3 processes exceeded the 70% survival criterion and were similar across processes and tumors.

Table 53. Survival Percentage of REP (TIL Final Product)

Because the number of fragments in each vial is less than the maximum required number, the estimated cell count on the collection day is calculated for each tumor. This assessment is based on the expectation that the clinical tumor is large enough on day 0 to be inoculated with 2 or 3 vials.

Table 54. Extrapolation of estimated cell counts to full-size 2 and 3 bottles for the Gen 3 process.

Immunophenotypic Analysis – Comparison of Phenotypic Markers in TIL End Products. Three tumor types, L4054, L4055, and M1085T, underwent TIL amplification during both Gen 2 and Gen 3 processes. Following collection, REP TIL end products were analyzed by flow cytometry to test for purity, differentiation, and memory markers. For all conditions, the percentage of TCR a/b+ cells exceeded 90%.

TILs collected from the Gen 3 process showed higher expression of CD8 and CD28 compared to those collected from the Gen 2 process. The Gen 2 process showed a higher percentage of CD4+.

TILs collected from the Gen 3 process showed higher expression of the central memory chamber compared to TILs collected from the Gen 2 process.

Activation and depletion biomarkers were analyzed in TILs from two tumors, L4054 and L4055, to compare the final TIL products from the Gen 2 and Gen 3 TIL amplification processes. Activation and depletion biomarkers for the Gen 2 and Gen 3 processes were similar.

Interferon-γ secretion upon restimulation. On the collection days, day 22 of Gen 2 and day 16 of Gen 3, TILs of L4054 and L4055 were restimulated overnight using coated anti-CD3 plates. M1085T was restimulated using anti-CD3, CD28, and CD137 beads. Under all conditions, the supernatant was collected and frozen 24 hours after restimulation. IFNγ analysis by ELISA was performed simultaneously on the supernatants from both processes using the same ELISA plate. Higher IFNγ production from the Gen 3 process was observed in all three tumors analyzed.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption during Gen 2 versus Gen 3 processes, cell supernatants were collected on REP initiation, scale-up, and collection days for tumors L4054 and L4055. The amount of IL-2 in the cell culture supernatant was measured using a Quantitate ELISA kit from R&D. A general trend indicates that IL-2 concentrations remained higher during Gen 3 compared to Gen 2. This may be attributed to the higher IL-2 concentration at Gen 3 REP initiation (6000 IU/mL) and residual culture medium throughout the process.

Metabolic substrate and metabolite analysis. The levels of metabolic substrates (e.g., D-glucose and L-glutamine) are measured as substitutes for overall culture medium consumption. Their reciprocal metabolites, such as lactate and ammonia, are also measured. Glucose is a monosaccharide in the culture medium, used by mitochondria to produce energy in the form of ATP. When glucose is oxidized, lactate is produced (lactate is an ester of lactate). Lactate is produced in large quantities during the exponential growth phase of cells. High levels of lactate can negatively impact cell culture processes.

For Gen 2 and Gen 3 processes, used culture media from L4054 and L4055 were collected on REP start, scale-up, and collection days. Used culture media were collected on days 11, 16, and 22 of Gen 2, and on days 7, 11, and 16 of Gen 3. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

L-Glutamine is an unstable essential amino acid required in cell culture media formulations. Glutamine contains an amine, and this amide group allows for the transport and delivery of nitrogen into cells. When L-glutamine is oxidized, cells produce toxic ammonia byproducts. To counteract L-glutamine degradation, GlutaMax ^™ was supplemented into the culture medium during the Gen 2 and Gen 3 processes, as it is more stable in aqueous solution and does not spontaneously degrade. In both tumor lines, the Gen 3 group showed a decrease in L-glutamine and GlutaMax ^™ during the process, as well as an increase in ammonia throughout the REP. In the Gen 2 group, constant L-glutamine and GlutaMax ^™ concentrations were observed, along with a slight increase in ammonia production. For ammonia, the Gen 2 and Gen 3 processes were similar at the collection day, showing only slight differences in L-glutamine degradation.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats at L4054 and L4055 was measured using Flow-FISH technology during Gen 2 and Gen 3. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Gen 3 showed similar telomere lengths to Gen 2.

CD3 analysis. To determine the clonal diversity of cell products generated in each process, the collected TIL final products from L4054 and L4055 were sampled and analyzed by sequencing the CDR3 portion of the T cell receptor for clonal diversity analysis.

Table 55 shows a comparison of the percentage of unique CDR3 sequences shared on L4054 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 199 sequences with Gen 2 final products, and 97.07% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 55. Comparison of the common uCDR3 sequence between Gen 2 and Gen 3 processes on L4054.

Table 56 shows a comparison of the percentage of unique CDR3 sequences shared on L4055 in TIL-collected cell products between Gen 2 and Gen 3. Gen 3 shares 1833 sequences with Gen 2 final products, and 99.45% of the top 80% of unique CDR3 sequences in Gen 2 final products are shared with Gen 3 final products.

Table 56. Comparison of common uCDR3 sequences between Gen 2 and Gen 3 processes on L4055.

CM1 and CM2 culture media were prepared in advance without filtration and kept at 4°C until used for tumor L4055 for Gen 2 and Gen 3 processes.

For L4055 tumors, on the REP initiation day, the culture medium was heated at 37°C for 24 hours for use in the Gen 2 and Gen 3 processes.

LDH was not measured in the supernatant collected during the process.

M1085T TIL cells were counted using a K2 cellometer.

On the tumor M1085T, samples are unavailable, such as supernatant for metabolic analysis; TIL products for activation and depletion marker analysis, telomere length and CD3-TCR vb analysis.

Conclusion . This example compares three independent donor tumor tissues based on functional quality attributes, expanded phenotypic characterization of the Gen 2 and Gen 3 processes, and culture medium consumption.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

Assuming pre-REP collection is performed on day 11 instead of day 7 and REP collection is performed on day 22 instead of day 16, extrapolated cell numbers for the Gen 3 process were calculated. In both cases, Gen 3 showed closer TVC numbers compared to the Gen 2 process, indicating that early activation enhances TIL growth.

Extrapolating values from other flasks (2 or 3) in the Gen 3 process, assuming larger tumor sizes and the maximum number of fragments required for each process as described. It was observed that collections performed on day 16 of the Gen 3 process achieved similar TVC doses compared to day 22 of the Gen 2 process. This observation is significant and indicates that early activation of the culture may reduce TIL treatment time.

The pre-REP and REP expansion comparisons for Gen 2 and Gen 3 were evaluated based on the survival rates of the resulting total viable cell and total nucleated cell populations. There was no comparability between the TVC cell doses on the collection day for Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than that of Gen 2, approximately 40% of the total viable cells collected at collection time.

In terms of phenotypic characterization, higher CD8+ and CD28+ expression was observed in all three tumors during the Gen 3 process compared to the Gen 2 process.

Compared to the Gen 2 process, the Gen 3 process shows a slightly higher central memory compartment.

Although the Gen 3 process is shorter in duration, the Gen 2 and Gen 3 processes show similar activation and exhaustion markers.

In the three tumors analyzed, the IFNγ production on the final product of Gen 3 was 3-fold higher than that of Gen 2. This data suggests that the Gen 3 process produces a more potent and efficient TIL product compared to the Gen 2 process, which may be attributed to higher expression of CD8 and CD28 on Gen 3. Phenotypic characterization showed a positive trend in CD8+ and CD28+ expression in Gen 3 compared to the Gen 2 process in all three tumors.

The telomere lengths of the final TIL products from Gen 2 and Gen 3 are similar.

The glucose and lactate levels of the final products of Gen 2 and Gen 3 were similar, indicating that the nutrient levels on the culture medium were not affected during the Gen 3 process because no reduction removal was performed each day during the process and the overall culture medium volume was smaller compared to Gen 2.

Compared to the Gen 2 process, the processing time of the entire Gen 3 process is reduced by about half, which will significantly reduce the cost of goods sold (COG) of TIL products amplified through the Gen 3 process.

IL-2 consumption indicates the general trend of IL-2 consumption during the Gen 2 process, and that IL-2 is higher during the Gen 3 process due to the lack of removal of old culture medium.

CDR3 TCRab sequence analysis revealed high clonal diversity in the Gen 3 process.

Adding feeder cells and OKT-3 on day 0 of the pre-REP allows for early activation of TILs and enables TIL growth using the Gen 3 process.

Table 57 describes the various implementation schemes and results of the Gen 3 process compared to the current Gen 2 process.

Table 57. Exemplary Gen 3 process characteristics.

Example 10: Exemplary GEN 3 process (also known as GEN 3.1)

This embodiment describes further studies regarding the comparability between Gen 2 and Gen 3 processes for TIL amplification. The Gen 3 process is modified to include an activation step early in the process, thereby increasing the final total live cells (TVC) output while maintaining phenotypic and functional profile. As described below, the Gen 3 implementation is modified to another implementation, referred to herein as Gen 3.1.

In some implementations, the Gen 3.1 TIL manufacturing process involves four operator interventions:

1. Tumor Fragment Isolation and Activation: On day 0 of the procedure, the tumor was dissected and final fragments of approximately 3 × ³ mm were generated (up to 240 fragments in total) and cultured in 1–4 G-REX100MCS culture flasks. Each flask contained up to 60 fragments, 500 mL of CM1 or DM1 medium, supplemented with 6,000 IU rhIL-2, 15 μg OKT3, and 2.5 × 10⁸ irradiated allogeneic mononuclear cells. The cultures were incubated at 37°C for 6–8 days.

2. TIL Culture Reactivation: On days 7-8, in both cases, replenish the culture by slowly adding CM2 or DM1 medium supplemented with 6,000 IU rhIL-2, 30 μg OKT3, and 5 × ^10⁸ irradiated allogeneic monocytes. Take care not to disturb the existing cells at the bottom of the culture flask. Incubate the culture at 37°C for 3-4 days.

3. Culture scaling up: Performed on days 10-11. During culture scaling up, in both cases, transfer all contents of the G-REX100MCS to a G-REX500MCS culture flask containing 4 L of CM4 or DM2 supplemented with 3,000 IU/mL IL-2. Incubate the culture flask at 37°C for 5-6 days until harvest.

4. Collection/Washing/Preparation: On days 16-17, reduce the volume of the culture flasks and combine them. Concentrate the cells and wash them with PlasmaLyte A pH 7.4 containing 1% HSA. Prepare the washed cell suspension with CryoStor10 at a 1:1 ratio and supplement with rhIL-2 to a final concentration of 300 IU/mL.

DPs are cryopreserved and stored in gaseous liquid nitrogen via controlled-rate freezing. *Complete standard TIL media 1, 2, or 4 (CM1, CM2, CM4) can be used instead of CTS ^™ OpTmizer ^™ T cell serum-free expansion medium, also known as the defining medium (DM1 or DM2), as mentioned above.

Procedure Description. On day 0, the tumor was washed three times and then fragmented into 3×3×3 final fragments. After fragmenting the entire tumor, the final fragments were then equally randomized and divided into three pools. One randomized fragment pool was introduced into each group, with the same number of fragments added according to the three experimental matrices.

Throughout the TIL amplification process, tumor L4063 was amplified using standard culture medium, and tumor L4064 was amplified using a specific culture medium (CTS OpTmizer). The components of the culture media are described herein.

CM1 complete medium 1: RPMI + glutamine, supplemented with 2mM GlutaMax ^™ , 10% human AB serum, gentamicin (50ug/mL), and 2-mercaptoethanol (55uM). The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM2 complete medium 2: 50% CM1 medium + 50% AIM-V medium. The final medium preparation is supplemented with 6000 IU/mL IL-2.

CM4 complete medium 4: supplemented with AIM-V containing GlutaMax ^™ (2mM). The final medium formulation was supplemented with 3000 IU/mL IL-2.

The basal ^{medium for} T cell expansion is supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L).

DM1: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 6,000 IU/mL IL-2.

DM2: CTS ^™ OpTmizer ^™ T cell expansion basal medium supplemented with CTS ^™ OpTmizer ^™ T cell expansion supplement (26 mL/L), CTS ^™ immune cell SR (3%), and GlutaMax ^™ (2 mM). The final formulation was supplemented with 3,000 IU/mL IL-2.

All types of culture media used, i.e., complete (CM) and defined (DM) media, were prepared in advance and kept at 4°C until the day before use, and were preheated in an incubator at 37°C for up to 24 hours prior to the treatment day.

For both tumor types, TIL cultures were reactivated on day 7. For L4063, reactivation was performed on day 10, and for L4064, scale-up was performed on day 11. Both cultures were collected and cryopreserved on day 16.

Results achieved . Cell counts and survival percentages were determined during Gen 3.0 and Gen 3.1 procedures. Amplification under all conditions followed the details described in this example.

For each tumor, the fragments were divided into three equal pools. Due to the small size of the tumors, the maximum number of fragments per bottle could not be reached. Total viable cells and cell viability were assessed under each condition for three different procedures. Cell counts were determined as TVC at reactivation on day 7, TVC at scaling up on day 10 (L4064) or day 11 (L4063), and TVC at collection on day 16/17.

Cell counts on days 7 and 10/11 were considered as FIO. Folds of expansion were calculated by dividing the TVC at day 16/17 (when collected) by the TVC at day 7 (reactivation). To compare the three groups, the TVC at the collection day was divided by the number of fragments added to the culture on day 0 to calculate the average number of viable cells per fragment.

Cell counting and viability assays were performed on L4063 and L4064. On both tumors, the Gen3.1 assay produced more cells per fragment than the Gen 3.0 assay.

Total viable cell count and fold expansion; percentage of survival during the procedure. Percentage of survival under all conditions was obtained after reactivation, scaling up, and collection. Release criteria for survival percentage were compared to final TILs after collection on day 16/17. All conditions assessed exceeded the 70% survival criterion and were similar across all procedures and tumors.

Immunophenotyping analysis – Phenotypic characterization of TIL final products. Flow cytometry analysis of the final products was performed to test purity, differentiation, and memory markers. Under all conditions, the population percentages of TCRα/β, CD4+, and CD8+ cells were constant.

Extended phenotypic analysis of REP TILs was performed. In both tumors, TIL products showed a higher percentage of CD4+ cells under Gen 3.1 conditions compared to Gen 3.0, and a higher percentage of CD28+ cells from the CD8+ population under Gen 3.0 conditions compared to Gen 3.1 conditions under both conditions.

TILs collected from Gen 3.0 and Gen 3.1 processes showed phenotypic markers similar to CD27 and CD56 expression on CD4+ and CD8+ cells, and similar CD28 expression on CD4+-gated cell populations. Comparison of memory markers on TIL final products:

Frozen samples of TIL collected on day 16 were stained for analysis. The TIL memory state was similar in the Gen 3.0 and Gen 3.1 processes. Comparison of activation and depletion markers on the final TIL products:

The activation and depletion markers of gated Gen 3.0 and Gen 3.1 processes are similar in CD4+ and CD8+ cells.

Interferon-γ secretion after restimulation. For L4063 and L4064, collected TILs were restimulated overnight using coated anti-CD3 plates. In both tumors analyzed, higher IFNγ production from the Gen 3.1 process was observed compared to the Gen 3.0 process.

Measurement of IL-2 levels in culture medium. To compare IL-2 consumption levels under all conditions and processes, cell supernatants were collected and frozen at the start of reactivation on day 7, scale-up on day 10 (L4064)/day 11 (L4063), and collection on day 16/day 17. The supernatants were then thawed and analyzed. The amount of IL-2 in the cell culture supernatant was measured according to the manufacturer's protocol.

IL-2 consumption was similar throughout the entire Gen 3 and Gen 3.1 processes, evaluated under the same culture conditions. IL-2 concentrations (pg/mL) were analyzed in the used culture media collected from L4063 and L4064.

Metabolite analysis. For each condition, used culture supernatant was collected from L4063 and L4064 at day 7 for reactivation initiation, day 10 (L4064)/day 11 (L4063) for scaling up, and day 16/day 17 for collection. The concentrations of glucose, lactate, glutamine, GlutaMax ^™ , and ammonia in the supernatant were analyzed using a CEDEX bioanalyzer.

Compared to the complete medium (2 g/L), the glucose concentration in the determined medium was higher, at 4.5 g/L. Overall, the glucose concentration and consumption were similar for the Gen 3.0 and Gen 3.1 processes across each medium type.

An increase in lactate was observed, and the increase in lactate was similar under Gen 3.0 and Gen 3.1 conditions as well as in the two media (complete medium and deterministic medium) used for reactivation amplification.

In some cases, the standard basal medium contains 2 mM L-glutamine and is supplemented with 2 mM GlutaMax ^™ to compensate for the natural degradation of L-glutamine into L-glutamic acid and ammonia under culture conditions.

In some cases, the specified (serum-free) culture medium used does not contain L-glutamine compared to the basal medium and is supplemented only with GlutaMax ^™ at a final concentration of 2 mM. GlutaMax ^™ is a dipeptide of L-alanine and L-glutamine, which is more stable than L-glutamine in aqueous solution and does not spontaneously degrade into glutamate and ammonia. Instead, the dipeptide gradually dissociates into individual amino acids, thereby maintaining a low but sufficient concentration of L-glutamine to sustain stable cell growth.

In some cases, the concentrations of glutamine and GlutaMax ^™ decreased slightly on scale-up days but increased on collection days, reaching levels similar to or closer to those on reactivation days. For L4064, the concentrations of glutamine and GlutaMax ^™ showed a slight decrease at a similar rate under different conditions throughout the process.

The ammonia concentration was higher in the samples grown in the standard medium containing 2 mM glutamine + 2 mM GlutaMax ^™ compared to the samples grown in the defined medium containing 2 mM GlutaMax ^™ . Furthermore, as expected, a gradual increase or accumulation of ammonia was observed during cultivation. No difference in ammonia concentration was observed under the three different test conditions.

Telomere repeats were performed using Flow-FISH. The mean length of telomere repeats on L4063 and L4064 cells during the Gen 3 and Gen 3.1 processes was measured using Flow-FISH technology. The results of the determination of the relevant telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Telomere measurements were performed. Telomere lengths in the samples were compared with those in the control cell line (1301 leukemia). The control cell line was a tetraploid cell line with long, stable telomeres that allowed for the calculation of relative telomere lengths. Similar telomere lengths were observed in the Gen 3 and Gen 3.1 processes evaluated in both tumors.

TCR Vβ lineage analysis

To determine the clonal diversity of cell products generated in each process, TIL end products were analyzed for clonal diversity analysis by sequencing the CDR3 portion of the T cell receptor.

Compare three parameters across three conditions:

● Diversity index of unique CDR3 (uCDR3)

● Total uCDR3 percentage

●For the first 80% of uCDR3:

○ Compare the total percentage of uCDR3 copies

○ Compare the frequency of unique clones

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 975 sequences, which is equivalent to 88% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

Compared with the Gen 3.1 test, the percentage of shared unique CDR3 sequences on TIL-collected cell products: The final products of the Gen 3 and Gen 3.1 tests contained a total of 2163 sequences, which is equivalent to 87% of the first 80% of unique CDR3 sequences in Gen 3 being shared with Gen 3.1.

The number of unique CD3 sequences identified from 1 × ^10⁶ cells collected on day 16 for different processes. Based on the number of unique peptide CDRs within the sample, the Gen 3.1 test conditions showed slightly higher clonal diversity compared to Gen 3.0.

The Shannon entropy diversity index is a reliable and commonly used comparative measure because the Gen 3.1 condition showed slightly higher diversity in both tumors compared to the Gen 3 process, indicating that the TCRVβ lineage of the Gen 3.1 test condition was more polyclonal than that of the Gen 3.0 process.

Furthermore, for tumors L4063 and L4064, the TCR Vβ lineage under the Gen 3.1 test conditions showed more than 87% overlap with the corresponding lineage of the Gen 3.0 process.

On the reactivation day, the IL-2 concentration on the used medium for the Gen 3.1 test of L4064 was lower than expected (similar to the Gen 3.1 control and Gen 3.0 conditions).

The low value may be attributed to pipetting error, but since the sample size was very small, it was impossible to repeat the measurement.

Conclusion . Compared with the Gen 3.0 and Gen 3.1 controls, the Gen 3.1 test conditions, which included feeder cells and OKT-3 on day 0, showed a higher TVC at day 16 collection. The TVC of the final product under the Gen 3.1 test conditions was approximately 2.5 times higher than that under Gen 3.0.

For the two tumor samples tested, the Gen 3.1 test conditions, with the addition of OKT-3 and feeder cells on day 0, reached the maximum capacity of the culture flasks at collection. Under these conditions, if a maximum of four flasks were started on day 0, the final cell dose could be between 80 and 100 × ^10⁹ TILs.

Maintain all quality attributes, such as phenotypic characterization, including the purity, depletion, activation, and memory markers of the final TIL product, between the Gen 3.1 test and the Gen 3.0 process.

In both tumor types analyzed, the final TIL product IFN-γ production in Gen 3.1 with added feeder cells and OKT-3 on day 0 was 3-fold higher than that in Gen 3.0, indicating that the Gen 3.1 process produces efficient TIL products.

No differences in glucose or lactate levels were observed under any of the test conditions. No differences in glutamine and ammonia were observed between the Gen 3.0 and Gen 3.1 processes under any of the culture medium conditions. Lower glutamine levels in the culture medium did not limit cell growth, indicating that the addition of GlutaMax ^™ alone to the culture medium is sufficient to provide the nutrients required for cell proliferation.

Scaling up was performed on days 11 and 10, respectively, and no significant difference was observed in the number of cells reached on the process's collection day. Furthermore, metabolite consumption was similar throughout the process in both cases. These observations suggest that the Gen 3.0 optimized process offers flexibility in terms of processing days, thereby promoting flexibility in manufacturing schedules.

As measured by CDR3 TCRab sequence analysis, the Gen 3.1 process, which added feeder cells and OKT-3 on day 0, showed higher clonal diversity compared to Gen 3.0.

Figure 32 illustrates the implementation scheme of the Gen 3 process (Gen 3 optimization process). Standard medium and serum-free CTS Optimizer medium can be used for TIL amplification in the Gen 3 optimization process. When using serum-free CTS Optimizer medium, it is recommended to increase the final concentration of GlutaMax ^™ on the medium to 4 mM.

Example 11: PD-1 gene knockout in TIL using TALEN

The embodiment described above illustrates the use of TALEN to generate PD-1 gene knockout (KO) TILs. In this embodiment, the general procedure is as follows:

Day 0 - Pre-REP

Day 7 - Activation

Day 12 - Electroporation

Day 13 - REP

Day 18 - Scale Expansion

Day 22 - Collection

Overview : This study used genetic engineering techniques to permanently knock out the PDCD-1 gene (PD-1 surface protein) using an mRNA construct. PD-1KO TILs were generated from one lung (L4256) and two melanoma (M1207 and M1209) tumors and then characterized.

Background : Binding between immune checkpoint receptors and their ligands has been shown to suppress T cell function. PD-1, a key inhibitory regulator expressed by activated/depleted T cells, has been extensively studied and is a major target for immunotherapy. Given the success of anti-PD-1 therapy in improving clinical outcomes in cancer patients, approaches targeting the PD-1/PD-L1 axis could enhance the efficiency of immunotherapy. Adoptive TIL therapy is a salvage immunotherapy for cancer, with an objective response rate of 56% in melanoma patients. Since PD-1 is regulated in TILs in response to antigen stimulation in vivo, the interaction between PD-1 and PD-L1 expressed by tumor cells may suppress TIL function. Gene editing of PD-1 has demonstrated high specificity for permanent gene disruption in human and mouse T cells. In the B16 mouse model, adoptive transfer of PD-1 T cells revealed superior tumor control and enhanced effector function and proliferative capacity of tumor-site infiltrating T cells. Therefore, this study examines whether PD-1 KO-mediated TILs can enhance TIL function in vivo after adoptive transfer.

Experimental design : Clinical manufacturing scale operations were performed as shown in the experimental layout in Figure 34, and an overview of the clinical manufacturing PD-1KO TIL process is provided in Tables 42 and 43.

The method is also applicable to the generation of PD-1/CTLA-4 dual-gene knockout TILs, PD-1/LAG-3 dual-gene knockout TILs, PD-1/CISH dual-gene knockout TILs, PD-1/TIGIT dual-gene knockout TILs, and PD-1/CBL-B dual-gene knockout TILs. In such cases, during the electroporation step, dual-gene knockout TILs can be generated by electroporating TILs with PD-1 and CTLA-4 TALEN mRNA, PD-1 and LAG-3 TALEN mRNA, PD-1 and CISH TALEN mRNA, PD-1 and TIGIT TALEN mRNA, or PD-1 and CBL-B TALEN mRNA, respectively.

Table 42: Outline of Pre-REP, Activation, Electroporation, and Resting Processes (Part I)

Table 43: Outline of REP, Scale-up, and Collection Process (Part II)

Results : Table 44 specifies the acceptance criteria used to evaluate the full-scale experimental efficacy, Table 45 specifies the additional end-product characterization tests performed, and Table 46 lists the tumors and related histories used in this study.

Table 44: In-process and Collected Product Release Testing and Acceptance Standards

Table 45: Characterization of the final product

Table 46: Tumors used in this study

On day 5, the total live cell count (TVC) after pre-REP yielded an average of 45 × ^10⁶ cells, with a mean survival rate of 73%. Cell doubling after activation was consistent across all three tumor types. Melanoma and the cervical indication both showed similar activation status, with activation markers of >50% CD25+ and CD71+. However, the lung tumor L4213 showed lower CD71 expression, at 33%. Ki67 expression was consistently greater than 90% in all three samples, indicating active cell proliferation. Table 47 summarizes the pre-REP and post-activation output for the three tumor types used in the study.

Table 47: Summary of Pre-REP and Post-Activation Cell Culture

parameter	Acceptance Standards	L4256	M1207	M1209
					Number of segments	≤100 segments	73	40	95
<![CDATA[TVC(×10<sup>6</sup>)]]>	≥5	26.9	13.4	69.2
					Survival rate (%)	N/A	97.9	88.6	97.5
<![CDATA[TVC(×10<sup>6</sup>)]]>	N/A	120.0	37.8	419.0
					Survival rate (%)	N/A	92.0	82.6	92.8
Double (D7 to D12)	N/A	2.2	1.5	2.6
					CD45+ / CD3+%	N/A	80.4	83.1	60.1
<![CDATA[CD25+%<sup>1</sup>]]>	N/A	92.2	88.5	66.8
					<![CDATA[CD71+%<sup>1</sup>]]>	N/A	75.6	87.8	48.9
<![CDATA[Ki-67+%<sup>2</sup>]]>	N/A	78.6	81.0	7.3

^1. Gating strategy of the group after activation on day 10: Lymphocytes > SSC single peak > FSC single peak > Live cells > CD3+CD45 > CD25+/CD71+

^2. Ki67 group's gating strategy: Lymphocytes > SSC unimodal > FSC unimodal > Live cells > CD45+ > Ki67+

On day 13 (after rest), survival rate and TVC recovery rate were calculated to determine the efficiency of electroporation. For all conditions, the recovery rate of the percentage of cells surviving after electroporation ranged from 82% to 92%. In electroporated samples, the TVC recovery rate ranged from 16% to 30%. Data from electroporation and resting are summarized in Table 48.

Table 48: Summary of Electroporation and REP Initiation

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The recovery rate is calculated by dividing the TVC or survival rate after electroporation by the TVC or survival rate before electroporation and then multiplying by 100.

^2. The TVCs injected into the REP were the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

The same number of cells were used to initiate the REP under both experimental and control conditions. As the REP scaled up, the average cell doubling occurred 4 times under all experimental and control conditions. At REP collection, TVC yield in the TriLink experimental group was similar to that in the simulated control group (TVC difference ≤20%). Non-electroporated TVC yield was slightly higher than that in the TriLink and simulated control groups. An average of 9.8 cell doublings was observed between day 11 and day 20.

The final product doses of the TriLink group were 16.5, 28.5, and 28.7 × ^10⁹ , respectively, with >90% cell viability and >98% CD45+CD3+ cell count. The final product was a highly enriched TIL product.

The data from the REP initiation and collection are summarized in Table 49.

Table 49: Post-REP Summary

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. The TVC inoculated into the REP was the lowest among all D11 TVCs for each tumor to maintain consistency across all conditions.

^2. The recovery rate is calculated by dividing the TVC after LOVO by the TVC before LOVO and multiplying by 100.

^3. Assuming all cells proceed to the hypothetical yield in both experimental and control conditions. On day 12 (before electroporation), cells are allocated for experimental (TriLink mRNA) and control conditions (simulated). Hypothetical yield is calculated by multiplying the collection by 3.

^4. Cell doubling is calculated using the formula “＝LOG(TVC on day 22 / TVC on day 11) /LOG(2)”.

^5. LOVO did not follow the plan.

^{The six} batches of materials were small in scale and did not undergo LOVO testing.

All TriLink mRNAs met the acceptance criteria.

Data on the gene knockout efficiency of PD-1KO TIL products evaluated are summarized in Table 50.

Table 50: TIL PD-1KO Summary

Data evaluating the TIL function of the collected samples (including IFNg, granzyme-B, and CD107a release output) are summarized in Table 51.

Table 51: Summary of TIL Functions

T = TriLink M = Mimic (electroporation in the absence of TALEN mRNA)

^1. Stimulation gating targeting live cells/CD3+

The functionality of TILs was characterized based on overnight stimulation of the final product with anti-CD3/anti-CD28/anti-CD137 Dynabeads. Supernatants were collected and frozen 24 hours after stimulation. ELISA was performed to assess the concentrations of IFNg and granzyme B released into the supernatant. IFNg release was greater than 500 pg/mL, and all TIL cultures secreted high levels (15036 pg/mL–40185 pg/mL) of granzyme B after stimulation.

CD107a (LAMP-1) is a marker of lymphocyte degranulation and is associated with cytotoxic activity following antigen or polyclonal stimulation (Aktas E et al., 2009). Flow cytometry was used to analyze the cell surface expression of CD107a in response to PMA stimulation for 2 hours in both CD4 ⁺ and CD8 ⁺ cells. The results presented in Table 51 compare CD107A expression measurements with those under unstimulated conditions. Similar to the TIL products generated in previous studies, most of the TILs in the final product expressed CD107A upon stimulation with PMA/IO (CD4+ and CD8+ TILs).

Multicolor flow cytometry was used to characterize TIL purity, identity, and the memory subset of REP TILs. ≤1% of detectable B cells, monocytes, or NK cells were present in the final collected TILs (Table 11). REP TILs were primarily composed of TCRa/b cells with effector memory differentiation. The CD4/CD8 ratio varied between samples but was consistent across different conditions within a single sample. No significant differences were observed between experimental and control conditions.

Data showing TIL purity, identity, and memory phenotype are summarized in Table 52.

Table 52: Characterization of TIL purity, identity, and memory phenotype

Note: The gating strategy for TIL purity is as follows:

Monocytes: viable cells%, CD14+

NK (Natural Killer) cells: viable percentage, CD14-, CD3-, CD56+CD16+

B cells: viable percentage, CD14-, CD3-, CD19+

T-CM-Central Memory T Cells

T-EM-effect memory T cells

T-EFF/TEMRA-effect memory and RA+ effect memory

Data showing TIL activation and TIL depletion levels for CD4+, CD8+, and CD3+ are summarized in Tables 53-55.

Table 53: Activation and Depletion States of CD4+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD4+ > CD4+ frequency %

Table 54: Activation and Exhaustion States of CD8+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD8+ > CD8+ frequency %

The CD4 and CD8 differentiation, activation, and depletion states under the TriLink mRNA condition were comparable to those under the control condition.

Table 55: Activation and Depletion States of CD3+TIL

T = TriLink M = Mimic (electroporation without TALEN mRNA) NE = Non-electroporation

The historical values (n=64) reported in Table 10 ^above are taken from the analysis of the TIL-2 group in REP-0112 - extended phenotypic characterization of lifileucel and correlation analysis between phenotype and clinical response.

^The historical range gating strategies and levels are as follows: FSC single peak > SSC single peak 2 > live cells > CD3+ > CD3+ frequency %

The activation and depletion status of TriLink mRNA in the CD3+ population were comparable to those in the control population.

Conclusion : The PD-1KO TIL process was developed at clinical manufacturing scale to amplify PD-1KO TIL to >16e9 within 20 days. All three batches of TriLink mRNA used at development scale met the acceptance criteria for release parameters.

All quality properties of TILs generated using the PD-1-KO TIL process, such as phenotypic characterization (including purity, memory, activation, exhaustion markers, and function), are comparable to those of melanoma Gen 2. See Table 56.

In summary, this work demonstrates that greater than 50% PD-1KO efficiency can be achieved in the clinical manufacturing of PD-1KO TILs using TriLink mRNA, thus supporting the use of TriLink mRNA in clinical manufacturing.

Table 56: Summary Table

The alternative PD-1 gene knockout (KO) TIL process is shown in Figure 35 and Table 57.

Table 57: Overview of the alternative full-scale process for PD-1 TIL culture

Example 12: Preclinical activity and manufacturing feasibility of gene-modified PDCD-1 gene knockout (KO) tumor-infiltrating lymphocyte (TIL) cell therapy

background

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TILs; lifileucel, LN-145) has demonstrated encouraging efficacy and safety in both ICI-injected and ICI-naïve settings in patients with advanced solid tumors. A single-dose lifileucel TIL cell therapy achieved a durable response in a post-ICI setting in patients with advanced/unresectable melanoma (Sarnaik AA, Hamid A, Khushalani NI et al., J Clin Oncol. 2021; 39(24):2656-2666; Larkin JMG, Sarnaik A, Chesney JA et al., J Clin Oncol. 2021; 39(Supplement 15): Abstract 9505.), with an objective response rate (ORR) of 36%, and the duration of response (DOR) was not reached after 33.1 months of follow-up. In patients with advanced melanoma who have not received ICI treatment, early combination therapy with lifileucel and pembrolizumab resulted in an ORR of 60% and a complete response rate of 30% (O'Malley D, Lee S, Psyrri A et al., J Immunother Cancer. 2021; 9(Supplement 2): Abstract 492).

Despite the effectiveness of anti-programmed cell death protein (PD)-1 ICI therapy, it is limited by poor tumor penetration, internalization, and endocytic clearance (Thurber GM, Schmidt MM, Dane Wittrup K. Adv Drug Deliv Rev. 2008; 60(12):1421-1434; Less JR, Posner MC, Boucher Y, Borochovitz D, Wolmark N, Jain RK. Cancer Res. 1992; 52(22):6371-6374; Netti PA, Baxter LT, Boucher Y, Skalak R, Jain RK. Cancer Res. 1995: 55(22): 5451-5458; Bordeau BM, Balthasar JP. Cancer Biol Med. 2021; 18(3): 649-664; Saga T, Neumann RD, Heya T et al., Proc Natl Acad Sci U SA. 1995; 92(19): 8999-9003); In contrast, TILs are actively transported into the tumor, can move independently by chemitraction, and are not cleared or internalized (Restifo NP, Dudley ME, Rosenberg SA. Nat Rev Immunol. 2018; 12(4): 269-281). Administration of anti-PD-1 antibodies is associated with immune-related adverse events (AEs) due to nonspecific upregulation of immune pathways via systemic activation and proliferation of T cells (Postow MA, Sildow R, Hellmann MD. N Engl J Med. 2018; 378:158-168).

IOV-4001 is a PDCD-1 gene knockout (KO) TIL cell therapy that enhances the efficacy of TIL cell therapy and eliminates the need for systemic anti-PD-1 therapy, while avoiding short-term and long-term systemic adverse events associated with anti-PD-1/PD-L1 therapy. Transcription activator-like effector endonucleases (TALENs) are hybrid molecules composed of a DNA-binding domain and a FokI nuclease. Combination of the two TALEN arms of the PDCD-1 gene encoding PD-1 mediates DNA double-strand breaks, leading to gene damage and PD-1 inactivation (Gautron AS, Juillerat A, Guyot V et al., Mol Ther Nucleic Acids. 2017; 9:312-321; Menger L, Sledzinska A, Bergerhoff K et al., Cancer Res. 2016; 76:2087-2093; Qasim W, Zhan H, Samarasinghe S et al., Sci Transl Med. 2017; 9(374):eaaj2013). A process has been established to generate TALEN-mediated PDCD-1KO TILs and amplify them to a treatment-relevant number with potent effector function and phenotypic markers indicating functional TILs (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; Sep 19–21, 2020. Abstract 1052p).

This embodiment describes the (1) preclinical activity of IOV-4001 in vivo, (2) clinical-scale manufacturing process development, and (3) phenotypic and functional characterization.

method

In summary, hIL-2NOG mice transplanted with melanoma tumor cells received autologous PD-1KO TILs (generated using an mRNA construct), TIL-mimicking (electroporation without TALEN), TIL-mimicking plus anti-PD-1 antibody adoptive transfer, or no adoptive transfer (n=14 each). Tumor size was measured twice weekly for 39 days. A 22-day clinical-scale manufacturing process was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, for the generation of PD-1KO TILs. The final PD-1KO TIL product was characterized by total live cells (TVC), purity (survival percentage), identity (CD45 ⁺ CD3 ⁺ %), and function (PD-1KO efficiency).

manufacture :

A 22-day clinical-scale manufacturing process for IOV-4001 was established, including a pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, to generate PDCD-1KO TILs (using an mRNA construct). Development operations were performed at Iovance Process Development (Tampa, FL) on a non-Good Manufacturing Practice (GMP) scale. Manufacturing operations were performed at a contract manufacturing organization (CMO) on a GMP scale.

In vivo antitumor activity :

Mice expressing human interleukin-2 (hIL2) under the control of the cytomegalovirus promoter (hIL-2NOG)15 were transplanted with melanoma tumor cells and received (A) autologous PDCD-1KO TIL, (B) TIL mimicry (TIL electroporation without TALEN), (C) TIL mimicry plus anti-PD-1 antibody, or (D) TIL-free adoptive transfer (n=14 each). Tumor size was measured twice a week for 39 days.

Product release :

The final PDCD-1KO TIL product was characterized as follows:

●Total viable cells (TVC) and purity (percentage of viable cells) were determined using an NC-200 ^™ (ChemoMetec, Denmark) automated cell counterstaining system via acridine orange/4',6-diamidindo-2-phenylindole (DAPI) counterstaining.

● Identity (CD45+CD3+ phenotype), analyzed by immunofluorescence staining and flow cytometry.

●Efficacy (interferon-γ [IFNγ] release), measured by ELISA using an IFNγ ELISA kit (R&D Systems, Minneapolis, MN, USA).

PDCD-1KO efficiency was evaluated by flow cytometry based on PD-1 expression in PDCD-1KO TILs compared with mimic TILs.

Phenotype :

Two multicolor flow cytometry teams were used to determine extended phenotypic markers of the final collected PDCD-1KO TIL products to characterize TIL purity, identity, memory subsets, activation, and depletion states.

Characterization :

IL-2 assay: To assess the safety of the PDCD-1KO TIL product, the in vitro proliferation capacity of the final product in the absence of IL-2 was evaluated over a 28-day period.

Karyotype determination: Cytogenetic examination was performed by NeoGenomics Laboratories (Fort Myers, FL, USA). Briefly, cryopreserved PDCD-1KO TIL samples were quiescent and activated to collect metaphase cells for G-banding cytogenetic analysis. Three replicates of mitotic cells were analyzed, fixed, and stained for G-banding.

Statistical analysis :

Unpaired Student's t-test was used to analyze phenotypic differences, and Wilcoxon's rank-sum test was used to detect differences in in vivo studies in mice; p < 0.05 was considered statistically significant.

result

In summary, for mice treated with PD-1KO TIL (6±2.8), mean ± SEM tumor size ( ^mm² ) at day 39 showed superior tumor control compared to simulated TIL (26±8.5, P<0.05), simulated TIL + anti-PD-1 (33±8.8, P<0.01), and no adoptive TIL metastasis (112±8.4, P<0.0001). Product properties from six clinical-scale manufacturing operations of PD-1KO TIL were acceptable, with median (range) TVC, purity, and identity of 8.3 × ^10⁹ (0.9 × ^10⁹ –35.7 × ^10⁹ ), 94% (91%–99%), and 99% (98%–99%), respectively. The median (range) PD-1KO efficiency was 48% (31%–84%). PD-1KO TILs exhibit comparable functions and phenotypes (differentiation, memory, activation, and exhaustion) to those of analog TILs.

As shown in Figure 38, enhanced in vivo antitumor activity was observed in mice treated with PDCD-1KO TIL compared to mice treated with TIL-mimicking alone or TIL-mimicking alone plus anti-PD-1 antibody.

As shown in Figure 39, all PDCD-1KO TIL products met the release criteria for dose, purity, identity, potency, and PDCD-1KO efficiency. No statistically significant differences were observed between the development and manufacturing processes. Both development and manufacturing processes produced final PDCD-1KO TIL products with comparable doses (A) and survival rates (B). The median (range) identity (CD45+CD3+%) of the final TIL products in the development and manufacturing processes was 98.5% (98%–100%) and 98.7% (96%–99%), respectively (C). The median IFNγ release in the development and manufacturing processes of the final PDCD-1KO TIL products was 4015 pg/mL and 4725 pg/mL, respectively (D). The median (range) PDCD-1KO efficiencies in the development and manufacturing processes were 63% (48%–81%) and 62% (31%–91%), respectively (E). The PDCD-1KO TIL product is comparable to the simulated TIL in terms of growth, purity, identity, and potency. As previously shown (Ritthipichai K, Machlin M, Lakshmipathi S et al., Presented at the ESMO Virtual Congress; September 19–21, 2020. Abstract 1052p), the dose, purity, identity, and potency results between the simulated and PDCD-1KO were comparable in development operations (data not shown).

As shown in Figure 40, the CD28 biomarker was highly expressed in both PDCD-1KO and simulated TILs in the development and manufacturing samples, while other biomarkers such as CD27, CD57, and KLRG1 were expressed at low levels (A). Subsets of naive (TN), central memory (TCM), effector memory (TEM), and effector memory RA+ (TEMRA) T cells were defined using CD45RA and CCR7 expression. Most TIL batches primarily exhibited the effector memory phenotype (B). No statistically significant differences in TIL differentiation biomarkers or memory phenotypes were observed between PDCD-1KO and simulated TILs during the development and manufacturing operations.

As shown in Figure 41, multicolor flow cytometry was used to characterize TIL activation and inhibitory receptor expression on CD4+ (A) and CD8+ TILs (B). No statistically significant differences in biomarker expression were observed between PDCD-1KO and simulated TILs during development and manufacturing operations.

As shown in Figure 42, the IL-2-independent proliferation assay demonstrated that no PDCD-1KO TIL products underwent malignant transformation after TALEN-mediated genome editing. In the absence of IL-2 (A and B), neither stimulated (anti-CD3/CD28) nor unstimulated samples proliferated. All samples cultured in the presence of IL-2 showed proliferation (A and B).

The karyotype analysis results from the PDCD-1KO TIL products are summarized in Figure 43. No clonal chromosomal abnormalities were observed in the G-banding analysis, indicating no genotoxicity following TALEN-mediated PDCD-1 genome editing. All samples analyzed by G-banding generated sufficient metaphase cells for a comprehensive study; normal G-banding patterns were observed.

in conclusion

In the presence or absence of anti-PD-1, PDCD-1KO TILs exhibited superior in vivo antitumor activity compared to mimic TILs (electroporation without TALEN), indicating that endogenous PD-1 inhibition can confer a functional advantage over antibody combinations. Clinical-scale manufacturing of PDCD-1KO TILs is feasible, and the quality properties and phenotype of the TIL products are acceptable. TIL properties were comparable across all development and manufacturing operations and met product release criteria. As determined by IL-2-independent proliferation assays, no malignant transformation occurred in PDCD-1KO TIL products following TALEN-mediated genome editing. No TALEN-induced clonal chromosomal abnormalities were identified by G-banding. Importantly, the absence of intact PDCD-1KO in the TIL products preserved other PD-1-dependent in vivo cellular functions. In conclusion, these data support the clinical investigation of IOV-4001 (an autologous PDCD-1KO TIL cell therapy) as a potential treatment option for patients with advanced solid tumors.

Example 13: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated, electroporated, and subjected to an 11-day REP process.

Pre-REP cells were stimulated for two days with plate-bound OKT3 (300 ng/ml) or TransAct (1:100), followed by electroporation of 5e6 cells resuspended in an electroporation buffer called “T buffer” in 4 mm spaced electroporation reaction cuvettes, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

After electroporation, cells were allowed to rest overnight at 30°C in CM1 medium containing IL-2, followed by REP. Simulated cells were seeded at different initial numbers (100kJ, 50kJ, 20kJ, 10kJ) and tests were performed to compare gene knockout efficiency.

result

Figures 44A and 44B show cell viability and recovery folds before electroporation. Specifically, Figure 44A shows cell viability after thawing compared to 2 days after stimulation with OKT3 or TransAct, and Figure 44B shows the recovery folds after thawing and stimulation with OKT3 or TransAct.

Figures 45A and 45B show the cell viability and recovery fold of cells after electroporation. Specifically, Figure 45A shows the recovery fold of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C, and Figure 45B shows the cell viability of cells stimulated with OKT3 or TransAct after overnight incubation at 30°C.

Figures 46A-46C show that, in the case of CD3+, CD8+, and CD4+ cells, TILs stimulated by TransAct exhibited lower KO efficiency compared to TILs stimulated by OKT3.

Example 14: Comparison of overnight rest at 30°C and 37°C

method

Pre-REP TIL lines (N=4) from different indications (head and neck, lung and breast) were thawed, activated and electroporated.

Pre-REP cells were stimulated with TransAct (1:100) for two days, followed by electroporation of 5e6 cells resuspended in T buffer in electroporation reaction cups with a 4 mm spacing, with 4 μg/1e6 cells of PD-1 TALEN in either the right or left arm.

Following electroporation, cells were seeded in equal numbers into 96-well plates and allowed to rest overnight at 30°C or 37°C in CM1 medium containing IL-2 or IL-15. Cells were then counted to determine the fold resuscitation and viability.

result

Figures 47A and 47B show cell viability and recovery folds after electroporation under all conditions (IL-2 or IL-15). Specifically, Figure 47A shows the recovery folds of cells after overnight rest at 30°C or 37°C, and Figure 47B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 48A and 48B show the cell viability and fold recovery rate of cells after electroporation when 6000 IU/mL IL-2 was used. Specifically, Figure 48A shows the fold recovery rate of cells after overnight rest at 30°C or 37°C, and Figure 48B shows the cell viability after overnight rest at 30°C or 37°C.

Figures 49A and 49B show cell viability and recovery folds after electroporation under various conditions. Specifically, Figure 49A shows the recovery folds of cells after overnight rest at 30°C or 37°C when using various concentrations of IL-2 or IL-15, and Figure 49B shows the cell viability after overnight rest at 30°C or 37°C when using different concentrations of IL-2 or IL-15.

Gene knockout efficiency was evaluated as described in Example 13. TILs were stimulated overnight with anti-CD3/CD28 beads at a ratio of 1 bead to 5 cells. Figures 50A-50C show that a decrease in KO efficiency was observed at the recommended 1:100 dilution under non-GMP TransAct stimulation.

Example 15: Comparison of TALEN mRNA concentration and incubation conditions

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed, activated, electroporated and subjected to a 10-day REP process.

Pre-REP cells were stimulated with OKT3 (300 ng/mL plate-bound) for two days, followed by electroporation of 1e6 cells resuspended in T buffer in electroporation reaction cuvettes with a 1 mm spacing. The right arm or left arm was given 4 μg, 2 μg, 1 μg, and 0.5 μg of PD-1 TALEN per 1e6 cells, respectively.

After electroporation, cells were seeded in the same number in 48-well plates and allowed to rest overnight in CM1 medium containing IL-2 at 30°C or 37°C, followed by REP.

result

Figures 51 and 52 show the cell viability and fold recovery rate of cells after electroporation with different concentrations of PD-1 TALEN mRNA. Specifically, Figure 51 shows the cell viability after electroporation with different concentrations of PD-1 TALEN mRNA, and Figure 52 shows the fold recovery rate after electroporation with different concentrations of PD-1 TALEN mRNA. No significant changes in viability or recovery rate were observed after electroporation with different concentrations of PD-1 TALEN mRNA.

Figures 53A and 53B show the cell viability and fold recovery rate when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C. Specifically, Figure 53A shows the fold recovery rate of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C, and Figure 53B shows the cell viability of cells cultured in media preheated to 30°C or 37°C overnight at 30°C or 37°C. No significant changes in viability or recovery rate were observed when cells were cultured overnight at 30°C in media preheated to 37°C or 30°C.

Gene knockout efficiency was evaluated as described in Example 13. Figures 54A-54C show the PD-1 KO efficiency in CD3+, CD8+, and CD4+ cells. The optimal KO efficiency of PD-1 TALEN was observed at 4 μg mRNA/million cells and overnight rest at 30°C. This concentration can be target-specific and also depends on the cell concentration used during electroporation. The concentration of TALEN mRNA used during the electroporation process did not appear to affect recovery rate and survival.

Example 16: Comparison of washing steps and centrifugation speed

method

Pre-REP TIL lines (N=4) from different indications (head and neck and lung) were thawed, activated (OKT3 plate binding 300 ng/mL), and subjected to washing steps that are usually performed before electroporation.

Pre-REP cells were stimulated for two days and then aliquoted into individual tubes at 5e6 cells per tube. They were washed with PBS, precipitated, washed with cell perforation buffer, precipitated again, and then resuspended in cell perforation buffer. Cells were resuspended in 500 μL of cell perforation buffer and counted by diluting the cells 1:10 in CM1. Cell counting was performed after each rotation to determine the percentage of cells lost during the washing step.

Repeat the procedure using different centrifugation speeds (400g, 300g, 200g, for 5 minutes) to improve cell recovery rate.

result

Figures 55A and 55B show the cell number (Figure 55A) and viability (Figure 55B) after various washing steps involving centrifugation at 400g for 5 minutes and rotation 3 times.

Figures 56A and 56B show the number of cells after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 57A and 57B show cell viability after washing with PBS or Cyto under rotation conditions of 400g, 300g, and 200g.

Figures 58A and 58B show the total number of cells and the total survival rate of cells after various rotation conditions, and Figure 59 shows the percentage of cells lost after various rotation conditions.

The results showed that washing with cell perforation buffer led to cell loss, and that the recovery rate may have been improved when cells were centrifuged in cell perforation buffer at 300g instead of 400g. Therefore, a single cell perforation buffer washing step is preferred.

Example 17: Comparison of OKT3 and TransAct stimuli

method

Pre-REP TIL lines (N=3) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates containing 300 ng/mL, TransAct 1:10 and TransAct 1:17.5, and then electroporated with PD-1 TAL.

result

Figures 60A-60C show the percentage loss and survival during electroporation using various stimulation methods, specifically the percentage of cell loss during the washing step (Figure 60A), the percentage of cell loss after electroporation (Figure 60B), and the cell survival rate after electroporation (Figure 60C). The results indicate that TransAct stimulation exhibits better recovery and survival rates after electroporation compared to OKT3.

Figures 61A-61C show the PD-1 gene knockout efficiency in CD3+ (Figure 61A), CD4+ (Figure 61B), and CD8+ (Figure 61C) cells using various stimulation methods. The results indicate that activation with OKT3 and TransAct leads to similar KO efficiency.

Figures 62A-62B show the cell viability (Figure 62A) and fold expansion (Figure 62B) of REP collections using various stimulation methods.

Example 18: Comparison of incubation temperatures after electroporation

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated for 2 days with OKT3 plates at 300 ng/mL, and then electroporated with PD-1 TAL.

After electroporation, cells were incubated overnight at different temperatures (25, 28, 30, 32, 35, 37 °C) and REP was performed the following day. Cell loss and viability, as well as PD-1 gene knockout efficiency, were evaluated after overnight incubation.

result

Figures 63A-63B show the percentage of cell loss (Figure 63A) and cell viability (Figure 63B) after electroporation at different incubation temperatures.

Figures 64A-64C show the gene knockout efficiency in CD3+ (Figure 63A), CD4+ (Figure 63B), and CD8+ (Figure 63C) cells using different incubation temperatures.

Figures 65A-65B show the fold expansion (Figure 65A) and cell viability (Figure 65B) of REP collections at different incubation temperatures.

Example 19: Comparison of Stimulus Day Timing Selection

method

Pre-REP TIL lines (N=2) from different indications (head and neck and breast) were thawed and activated with GMP TransAct 1:17.5 on different dates (days 0, 3, 5, and 7).

Electroporation of cells with PD-1 TAL was performed on days 9 and 12 to determine the optimal dates for stimulating pre-REP cells to maximize PD-1 KO efficiency and pre-REP cell number.

result

Figures 66A-66C show cell growth (Figure 66A), first electroporation gene knockout efficiency (Figure 66B), and second electroporation gene knockout efficiency (Figure 66C) of cells stimulated on different dates.

Figure 67 shows the percentage growth of cells stimulated on different dates at 3 days of rest.

Example 20: Overview of PD-1KO TIL Products

"PD-1KO TIL Product" is an autologous tumor-infiltrating lymphocyte (TIL) preparation. These lymphocytes have been genome-edited with a transcription activator-like effector nuclease (TALEN) to disrupt the gene for programmed cell death protein-1 (PD-1). The PD-1 TALEN is engineered to bind to a distinct DNA sequence adjacent to the nucleotide lysis target site on the PDCD1 gene. DNA binding leads to the dimerization of two separate FokI endonuclease domains, producing a functional DNA endonuclease that can generate a sequence-specific double-strand break (DSB) in exon 2 (the central protein-coding region of PDCD1). This DSB is repaired via endogenous error-prone DNA repair pathways (e.g., non-homologous end joining (NHEJ)), resulting in nucleotide insertions or deletions, thereby disrupting the protein-coding sequence of PD-1.

The 4-step PD-1KO TIL product manufacturing process is a modified version of the method used to produce the autologous TIL product lifileucel (IND 16317 and 16819). This process consists of the following steps: a first step of TIL growth in the presence of IL-2, followed by transient activation of T cells with TransAct (a bead-free colloidal polymer nanomatrix conjugated with humanized CD3 and CD28 agonists), electroporation of PD-1KO TALEN mRNA (left and right arms), overnight rest at 30°C, and a final rapid expansion protocol (REP) step.

Cross-study PD-1 KO TIL product characterization data (Table 58) and process development data indicate that changes in the manufacturing process do not affect product quality (characteristics, survival rate, PD-1 gene knockout efficiency) and result in an average PD-1 KO efficiency of approximately 60%. In the PDX mouse model system, its antitumor activity is superior to that of the simulated control TIL in the presence of pembrolizumab.

Table 58. List of Non-Clinical Studies

Abbreviations: GLP = Good Laboratory Practice; IL-2 = Interleukin-2; KO = Gene Knockout; OCA = Oligonucleotide Capture Assay; PD-1 = Programmed Cell Death Protein-1; PDCD1 = Gene of Programmed Cell Death Protein-1; PDX = Patient-Derived Xenograft; TALEN = Transcription Activator-like Effector Nuclease; TCR = T Cell Receptor; TIL = Tumor-Infiltrating Lymphocytes

¹ This study evaluates different batches of PD-1KO TIL products, some of which were evaluated as part of a GLP compliance study.

Overview

The non-clinical studies conducted support the safety of the PD-1KO TIL product initiating clinical studies through the following:

● Confirmed that PD-1 TALEN expression in TILs is transient after electroporation;

● Characterization using NGS confirmed that 19 of the top 20 potential off-target sites identified in the OCA analysis were free of TALEN-related mutagenesis. Minimal off-target activity was detected at one candidate site (Cand 3). This is unlikely to pose a safety risk to the product because of the low mutation frequency and the fact that the site corresponds to a non-coding RNA locus not known to be associated with the disease.

● No clonal chromosomal aberrations were identified in the karyotype analysis of the PD-1KO TIL drug product, therefore there is no evidence that TALEN-mediated genome editing has significant genotoxicity;

● No evidence of IL-2-independent proliferation was found, thus demonstrating that no malignant transformation was observed in any of the PD-1KO TIL drug products tested.

Furthermore, non-clinical studies support the potential antitumor activity and mechanism of action of PD-1KO TIL products based on the following observations:

● Reproducible high levels of TALEN-mediated PDCD1 mutagenesis (approximately 60% on average) were achieved in the genome of autologous TILs, with the PDCD1 mutagenesis rate correlated with decreased PD-1 expression. Therefore, PD-1KO TIL products constitute true PD-1KO gene therapy products.

● The general characteristics of autologous TILs, which are considered crucial for anti-tumor activity, have been shown to be unaffected or positively affected by PD-1KO, including survival, proliferative capacity, differentiation status, memory T cell subset, activation/exhaustion status, TCR Vβ diversity, T cell effector function, and cell lysis activity.

● Enhanced tumor burden control was observed in PDX mouse models treated with ACT and PD-1KO TIL products, compared with control groups that did not use ACT, ACT with unmodified autologous TIL, and ACT with unmodified autologous TIL combined with anti-PD-1 antibody.

In summary, these studies demonstrate that isolating, expanding, and genetically engineering PD-1 knockout T cells from various tumors is safe and exhibits potent antitumor activity in both in vitro and in vivo models. These observations suggest that PD-1KOTIL products possess the potential and manageable safety profile of an autoimmune therapy for treating patient populations constituting unmet medical needs.

result

Figures 68A-68C show the PD-1 gene knockout efficiency. Figure 68A shows the PD-1 KO efficiency determined by measuring PD-1 receptor expression on the PD-1 KO TIL product using flow cytometry. Figure 68B shows the PD-1 KO efficiency determined by measuring the total number of PDCD1 genes with insertions/deletions using NGS. Figure 68C shows the correlation between the PD-1 KO efficiency assessed by flow cytometry and the PDCD1 gene modifications determined using NGS.

Figure 69 shows that most of the insertions/deletions in PDCD1 after TALEN genome editing were nucleotide deletions.

Phenotypic characterization

The proliferative capacity, survival, and immunophenotype of PD-1KO TIL products relative to unmodified autologous TILs were evaluated using a study-scale process derived from five different types of tumors (breast (n=5), lung (n=2), HNSCC (n=2), melanoma (n=2), and ovary (n=1)).

Cell viability and proliferation capacity

The PD-1KO TIL product exhibited cell viability comparable to non-electroporated (NE) TILs, with an average viability of approximately 93% for both types of samples. The proliferative capacity of the PD-1KO TIL product was also comparable to that of NE TILs, with average fold increases of 1217 and 1565, respectively, during the REP step of the production process.

Differentiation status of PD-1KO TIL products and NE TIL

PD-1KO TILs exhibit a differentiation state comparable to NE TILs, as determined by similar expression levels of CD27, CD28, CD56, BTLA, and KLRG-1. Therefore, PD-1 inactivation does not alter the differentiation state of TILs.

Memory T cell subsets in PD-1KO TIL products and NE TIL

In this study, over 98% of PD-1KO TIL products and NE TILs were T effector cells (CD45RA-CCR7-), while _{the TCM} subset (CD45RA-CCR7+) accounted for less than 1% of the cells analyzed. Importantly, the proportion of terminally differentiated T cells was negligible.

Activation and depletion states in PD-1KO TIL products and NE TIL

As indicated by the expression levels of CD69, CD25, and DNAM, the activation state of the PD-1KO TIL product was relatively high and comparable to that of NE TIL. Consistent with its recent activation, both the PD-1KO TIL product and NE TIL expressed high levels of co-inhibitory molecules (TIGIT and TIM-3). These results suggest that the activation and depletion states are not altered by PD-1 inactivation.

In summary, PD-1 gene knockout did not significantly affect TIL survival, proliferation capacity, or immune phenotype, indicating that the activity of PD-1KO TIL products associated with these biological characteristics was similar to that observed in unmodified autologous TILs.

Figures 70A-70B show the distribution of TCR Vβ isotypes in the CD3+PD-1- subset within the main PD-1KO TIL products and NE TILs. Main TILs generated from lung tumor L4022 (Figure 70A) and breast tumor EP11017 (Figure 70B) were electroporated with PD-1 TALEN mRNA (PD-1KO) or not electroporated (NE). Twenty-four TCR Vβ families were evaluated by flow cytometry in the CD3+PD-1-T cell subset. The percentage of TCR Vβ isotypes is plotted in pie charts. All remaining isotypes not identified by flow cytometry are pooled and shown in light blue (other). The color represents each TCR Vβ isotype, as indicated in the legend below the pie chart.

Figures 71A-71B show the effector function of PD-1KO TILs as measured by MLR and multifunctionality. For Figure 71A, TILs were generated in duplicate from melanoma (n=3), breast cancer (n=1), and lung cancer (n=1) samples. The responsiveness of NE TILs (black circles) and PD-1KO TILs (white circles) to HLA-mismatched lymphocytes was tested. IFN-γ secretion from the supernatant was assessed using ELISA. The horizontal and vertical bars represent the mean and standard error, respectively. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance. For Figure 71B, TILs were generated from melanoma (n=1) and breast cancer (n=3) samples. The multifunctionality intensity index (PSI), a measure of the ability to produce multiple cytokines at the single-cell level, was measured in activated host PD-1KO and NE TILs. The results are shown as a bar graph of the mean and corresponding standard error of cytokines involved in the inflammatory (pink), regulatory (beige), chemoattractive (purple), stimulative (blue), and effector (green) pathways. Statistical significance was assessed by paired Student's t-test. ns = no statistical significance.

In vivo studies: Patient-derived xenograft (PDX) mouse model studies

This study compared the antitumor activity of PD-1KO TIL products with unmodified TILs in a patient-derived xenograft (PDX) model grown in hIL-2 transgenic mice. Paired autologous PDX tumor cell lines/TIL formulations derived from freshly excised human melanoma lesions (n=1; M1152) were used in this study. The PD-1KO efficiency level was 75%. No differences in cell viability, proliferation, differentiation, memory T cell subsets, or exhaustion status were observed between M1152 PD-1KO and simulated TILs.

Characterization of PDX-derived melanoma tumor cell lines

The M1152 PDX tumor cell line is a PDX tumor-derived gene from NSG mice implanted with melanoma tumor fragments. The PDX melanoma cell line was characterized by measuring melanoma tumor-specific markers called melanoma-associated chondroitin sulfate proteoglycan (MCSP) and PD-L1 expression via flow cytometry. Approximately 80% of the tumor cells expressed MCSP protein, indicating that the majority of these cells were of melanoma origin. IFN-γ treatment induced an upregulation of PD-L1 from 13% to 82% within 48 hours. This encapsulates an in vivo drug resistance mechanism in which tumor cells increase PD-L1 expression in response to T cell secretion of IFN-γ during tumor cell lysis and supports the relevance of the M1152 PDX model for assessing M1152 PD-1 KO TIL activity.

In vivo tumor burden control of M1152 PD-1KO TIL

To determine the effect of PD-1KO TILs on tumor growth, PD-1KO or TIL-mimicking cells were adoptively transferred into hIL-2NOG mice implanted with autologous melanoma cells. Combinations of anti-PD-1 and TIL-mimicking cells served as controls for PD-1/PD-L1 pathway blockade. The in vivo efficacy of PD-1KO TILs was assessed by tumor burden reduction.

Figure 72 shows the in vivo antitumor activity of the M1152 PD-1KO TIL product. hIL-2NOG mice (n=14 per treatment group) transplanted with melanoma tumor cells underwent adoptive metastasis with either PD-1KO or a TIL-mimicking agent. Anti-PD-1 antibody treatment in combination with a TIL-mimicking agent was included as a control for PD-1/PD-L1 blockade. Tumor volume was calculated as length × width and plotted as a function of time post-implantation. Statistical significance was assessed using the Wilcoxon rank-sum test, with *, **, and **** indicating p-values <0.05, <0.01, and <0.0001, respectively, and considered statistically significant.

Tumor reduction was observed in mice treated with PD-1KO and TIL-mimicking antibodies, with or without anti-PD-1 antibody therapy. This indicates that TILs can recognize and lyse transplanted tumor cells, thus validating the tumor-specific recognition of TILs via MHC and TCR conjugation. Furthermore, even in combination with anti-PD-1 therapy, PD-1KO TILs induced superior tumor control relative to TIL-mimicking TILs. The failure of anti-PD-1 to enhance the in vivo efficacy of TIL-mimicking TILs in immunodeficiency and the lack of primary and secondary lymphoid organs in hIL-2NOG mice suggests that the in vivo mechanism of systemic PD-1 suppression may require an intact immune system. Conversely, the improved antitumor activity of PD-1KO TILs compared to TIL-mimicking TILs indicates that endogenous PD-1 suppression confers a functional advantage on TILs.

Therefore, this study demonstrates that TALEN-mediated PD-1KO confers a functional advantage on TILs. This finding is consistent with several published studies that describe an enhanced ability of PD-1KO T cells to control tumor growth relative to unmodified T cells (Menger 2016, Marotte 2020).

Figures 73A-73B show the persistence of TALEN protein in autologous TILs as a function of time, as measured by Western blotting. TILs generated from melanoma (n=6; M1011, M1017, M1025, M1030, M1034, M1040) were electroporated with PD-1 TALEN mRNA. NE TILs were used as a baseline control. TALEN protein was detected in whole-cell extracts from TILs by Western blotting at 8, 10, 12, 14, and 18 hours post-electroporation (Figure 73A) and on days 1, 2, 4, and 6 (Figure 73B). The intensity of the TALEN-specific bands was quantified by densitometric analysis, with the resulting OD values averaged over time and background corrected for the mean values of NE TILs. The mean values are plotted as bars, and the standard error is shown as a vertical line.

Quantification of mutagenesis at target sites and candidate off-target sites in PD-1 TALEN

Information about each candidate off-target site is included in Table 59.

Table 59. Off-target site analysis

IL-2-independent proliferation assay

By day 28 of analysis, none of the PD-1KO TIL product samples evaluated using CD3/CD28 activation proliferated in the absence of IL-2. All samples cultured in the presence of IL-2 proliferated by day 28, with a minimum amplification of 1.7-fold and a maximum of 407-fold. Notably, paired TALEN-edited TILs and unmodified TILs isolated from the same tumor showed comparable overall amplification folds across samples. These results demonstrate the safety of the PD-1KO TIL drug product by confirming the absence of cell proliferation in the absence of IL-2, indicating no malignant transformation of cells in this product.

Cytogenetics/karyotype analysis

Two aberrant karyotypes were observed, with X chromosome monosomy detected in 4–5 of the 20 metaphase diffuses analyzed. These structural abnormalities were detected in both non-genome-edited TIL samples and TALEN-edited TIL samples, therefore, these abnormalities cannot be attributed to TALEN-mediated genome editing. Furthermore, as assessed in a cytogenetic analysis of 200 metaphase cells, no significant increase in mutagenesis was observed in PD-1 TALEN-edited TILs compared to unmodified TILs. Based on these results, it is concluded that no evidence of TALEN-induced clonal chromosomal aberrations was found in any of the tested samples, and therefore, no significant genotoxicity was identified following TALEN-mediated genome editing.

Example 21: Exemplary TIL Manufacturing Process

Figures 74A-F depict an exemplary TIL manufacturing process. Briefly, on day 0, tumor tissue is isolated from hypothermosol, the bioload sample is stored in transport medium, and tumor fragments are seeded into multiple (2, 3, or 4) G-Rex 100MCS culture flasks at a density ≤50 fragments/flask. Excess fragments are rapidly frozen. In some embodiments, the activation step can be incorporated into the pre-REP step, which provides a better co-stimulatory environment for TILs within the rosette/tumor ME. For example, on day 3, 60 μg of OKT3 or TransAct is added to each of the multiple G-Rex 100MCS culture flasks, and the cells undergo initial activation. On days 7/8, the volume is reduced, the sample is filtered, and transferred to a pooled EXP1000. The sample is removed for cell counting/viability analysis. Cells are washed, centrifuged at 400 g, 20 °C for 10 min, and aliquoted into TALEN and control samples at a ratio ≥9:1. Cells were resuspended in T buffer and electroporated at 10 × ^10⁶ cells/cup with TALEN mRNA (TALEN sample) or without RNA (control sample). Electroporation control and TALEN samples from each cup were seeded into G-Rex100M culture flasks containing 100 mL of medium (containing 6000 IU/mL IL-2) and incubated at 37°C for 1 hour. Feeder cells were irradiated, thawed, collected, and incubated with IL-2. Samples were removed for cell counting and viability analysis. Feeder cells and additional IL-2 and OKT3 were added to the incubated control and TALEN samples in G-Rex 100MCS culture flasks to generate REP cultures (1x G-Rex 100MCS for control REP culture, ≤9x G-Rex 100MCS for TALEN REP culture). On days 10/11, 500 mL of culture medium along with 3000 IU/mL IL-2 was added to each culture flask of the REP culture. Alternatively, the cell culture medium could be completely replaced with fresh medium. This step did not require transferring the cell suspension and did not use G-Rex 500MCS. On day 16, the volume was reduced, and the samples were collected. The samples were transferred via a blood filter, and the samples were removed for cell counting/viability analysis. Control samples were centrifuged, and the control final formulation was prepared and cryopreserved at a controlled rate. TALEN samples were processed using the LOVO system, and the TALEN residue final formulation was prepared and cryopreserved at a controlled rate. The prepared product samples were then subjected to quality control analysis.

This process has the following advantages: a) it eliminates the suspension transfer step for activation in the bag; b) it allows for 1/10 scale control; c) it eliminates in-process transfer to G-Rex10; d) it eliminates the overnight incubation step at 30°C, allowing for immediate reactivation (REP) of the medium at ambient temperature; and e) it eliminates one treatment day.

Example 22: A Phase 1/2 Study Evaluating the Efficacy and Safety of PD-1KO TIL Infusion

introduction

The phase 1/2 open-label study of PD-1 knockout tumor-infiltrating lymphocyte (PD-1KO TIL) products was conducted in participants with unresectable or metastatic melanoma or stage III or IV non-small cell lung cancer.

PD-1KO TIL product is an autologous TIL product that has undergone genome editing with transcription activator-like effector nucleases to disrupt the gene PDCD1 encoding PD-1. This protocol will evaluate PD-1KO TIL product as a treatment option for participants with unresectable or metastatic melanoma or stage III or IV NSCLC.

Research Principles

This study marks the first human investigation of the PD-1KO TIL product. The antitumor activity of the PD-1KO TIL product and its ability to directly target and kill tumor cells in a manner similar to non-genome-edited autologous TIL products were evaluated, with the potential for enhanced antitumor activity due to PDCD1 disruption.

Consistent with the hypothesis, enhanced antitumor activity of the PD-1KO TIL product was observed relative to non-genome-edited autologous TILs in a patient-derived xenograft mouse model. A proof-of-concept nonclinical study of the PD-1KO TIL product further demonstrated that TALEN-mediated PDCD1 mutagenesis was reproducible, correlated with reduced PD-1 cell surface expression, and indicated that the general biological characteristics of autologous TILs considered crucial for antitumor activity were either unaffected or positively affected by PD-1 gene knockout. In the absence of conventional nonclinical animal models associated with human TIL testing, in vitro studies supported the safety of the PD-1KO TIL product. Low off-target activity was detected at one of the top 20 potential off-target sites. Given the low mutation frequency and the fact that this site corresponds to a non-coding RNA not known to be associated with the disease, this is unlikely to pose a safety risk to the product. Additional in vitro studies showed no TALEN-related clonal chromosomal abnormalities following TALEN-mediated genome editing.

background

Unresectable or metastatic melanoma, as well as stage III and IV NSCLC, represent malignancies with a significant unmet medical need. Despite recent advances in melanoma treatment, including ICIs (e.g., anti-cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4] and anti-PD-1 monoclonal antibodies [mAb]) and targeted therapies (e.g., proto-oncogene B-Raf [BRAF] and mitogen-activated protein kinase [MEK] inhibitors), unresectable or metastatic melanoma remains difficult to treat and continues to be a major public health problem (Howlader 2020; Steininger 2021). While several effective NSCLC therapies are currently available, including ICIs (i.e., nivolumab and pembrolizumab), platinum-based double chemotherapy, and tyrosine kinase inhibitors (TKIs) or other targeted therapies against driver mutation-positive tumors, none have provided adequate activity and durable cancer control. Ultimately, most tumors progress despite the initial effectiveness of the available therapies. Therefore, despite the approval of ICI, there remains a significant unmet medical need for melanoma and NSCLC patients who have made progress on available standard care therapies.

Autologous TILs have demonstrated durable responses and have the potential to address a major unmet need in clinical trial participants with metastatic melanoma who have limited treatment options, including those with primary refractory to anti-PD-1/PD-L1 therapy (Rosenberg 2011; Larkin 2021; Sarnaik 2021). Similarly, preliminary data suggest that TILs elicit clinically meaningful responses in NSCLC participants who have experienced disease progression following a series of therapies, including ICIs, and in participants with epidermal growth factor receptor (EGFR) driver mutation-positive tumors (Creelan 2021; Schoenfeld 2021).

The antitumor activity of TILs can be inhibited through interactions between lymphocytes and the tumor microenvironment. Specifically, signaling that occurs when PD-1 on the surface of T cells binds to PD-L1 on tumor cells negatively impacts T cell antitumor activity. Consistent with this finding, various antibody therapies that specifically block the PD-1/PD-L1 pathway, such as the anti-PD-1 antibodies pembrolizumab and nivolumab, have shown potent activity in solid tumors, both as single agents and in combination with non-genome-edited autologous TILs. Notably, in addition to their promising anticancer activity, clinical studies of non-genome-edited autologous TIL therapy combined with anti-PD-1 antibodies in unresectable or metastatic melanoma, recurrent and/or metastatic head and neck squamous cell carcinoma, and persistent, recurrent, or metastatic cervical cancer provide preliminary evidence of the safety of PD-1 signaling inactivation in the context of adoptive cell therapy (ACT) with TILs, suggesting a safety profile consistent with that of individual therapies (O'Malley 2021). This is consistent with the results of other studies conducted to date in participants with metastatic melanoma (evaluating the combination of TIL therapy and immune checkpoint inhibition) (Besser 2013; Mullinax 2018) and a study investigating the administration of TIL therapy in combination with nivolumab in participants with NSCLC (Creelan 2021).

Systemic administration of anti-PD-1 antibodies generates adverse events (AEs) due to the non-specific upregulation of immune pathways via T cell activation and proliferation. As a potential alternative to this treatment, TALEN genome editing technology is used to specifically inactivate PDCD1 in autologous TILs, thereby producing PD-1KO TIL products, PD-1 gene knockout products. By limiting the inhibition of PD-1 signaling in therapeutic tumor-derived T cells, PD-1KO TIL product therapy is expected to provide a safer alternative to ICI therapy, which imparts systemic inhibition of the PD-1 pathway. Furthermore, compared to anti-PD-1 antibody regimens requiring repeated administration, PD-1KO TIL product therapy has the potential to offer clinical benefit as a one-time treatment.

Benefit/Risk Assessment

Currently, no clinical safety data are available for the PD-1KO TIL product, as this is the first human study of the PD-1KO TIL product. There are no clinical safety data from studies of ACT and autologous TILs with PD-1 gene knockout via genome editing; however, safety data are available from non-genome-edited TIL regimens administered alone or in combination with anti-PD-1 antibodies. Safety data from studies of non-genome-edited TIL products are considered relevant because they are manufactured using a derivative process of the PD-1KO TIL product manufacturing process, have the same formulation as the PD-1KO TIL product, and are administered as part of a regimen similar to the proposed PD-1KO TIL product regimen.

The risk and benefit assessment presented below is based on observations related to the administration of NMA-LD and IL-2, as reported in their respective prescribing information, and clinical experience with similar treatments to PD-1KO TIL products, specifically non-genome-edited autologous TIL products administered alone or in combination with anti-PD-1 antibodies, as part of a regimen similar to the proposed PD-1KO TIL product regimen.

Benefit assessment

Potential benefits to participants include the possibility of receiving a research intervention that could stop, slow, or reverse cancer progression in patient populations lacking effective treatment options.

Overall Benefits and Risks Conclusion

Considering the following, the benefit-risk profile of the PD-1KO TIL product treatment regimen is suitable for participants planned to be enrolled in the IOV-GM1-201 study:

●The known toxicities of non-genome-edited autologous TIL regimens (i.e., NMA-LD, IL-2, and non-genome-edited autologous TIL products) administered alone or in combination with anti-PD-1 antibodies are monitorable and manageable.

● Measures taken to minimize the risk to participants

● The lack of effective treatment options that provide durable benefits to the study population, and

●This new therapy has the potential to improve the antitumor activity and safety profile of existing TIL-based solid tumor treatment regimens and PD-1 inhibitor therapies.

Goals and End Points

Research Design

Overall Design

This is a phase 1/2, open-label, non-randomized, multi-cohort, multicenter study with a safety lead-in period, evaluating autologous TIL regimens using the PD-1KO TIL product in participants with unresectable or metastatic melanoma (cohort 1) or stage III or IV NSCLC (cohort 2). In each cohort, tumor samples were excised from each participant and cultured in vitro to produce IOV-4001. Participants were infused with the PD-1KO TIL product, followed by IL-2, after lymphocyte depletion chemotherapy including cyclophosphamide and fludarabine. During the safety lead-in period, the first three participants from either cohort will receive treatment in a staggered manner. Once each participant successfully completes the 28-day DLT observation period (starting at the time of PD-1KO TIL product infusion), the next participant may be authorized to continue with NMA-LD. Subsequent recruitment will follow a standard 3+3 downgrade design, allowing additional participants to join while evaluating the safety and tolerability data of the emerging PD-1KO TIL product. These safety data will be reviewed by the SRC, which comprises representatives of the sponsor and researchers from all recruited DLT-evaluable participants, to advise on dose level decisions. Overall safety will be monitored by an independent DSMB. Recruitment for both cohorts will continue after the first 3 to 6 participants have completed the DLT observation period and received DSMB approval; there will be no staggered periods between participants.

Scientific Principles of Research Design

The study was designed without a control group because these participants, who had undergone extensive pretreatment, had no alternative effective treatment options that could provide durable benefits to the study population. Although no comparisons were available, given the nature of the participants' advanced, refractory disease and the fact that the study intervention (autologous TIL regimen) was administered as a single, one-time therapy, any major tumor regression observed during the study could be presumed to be attributable to the study intervention.

The primary efficacy endpoint of the second phase of this study, namely the ORR according to RECIST v1.1, is the validated method for evaluating tumor response in a single-arm study.

Study population

Prospective approval of deviations from recruitment criteria is not permitted; this is also known as program waiver or exemption.

Key inclusion criteria

Participants are eligible to be included in the study only if they meet all of the following criteria:

1. Participants must be at least 18 years old when signing the informed consent form.

2. Participants who were histologically or pathologically diagnosed with unresectable or metastatic melanoma of stage IIIC, IIID, or IV (cohort 1) or stage III or IV NSCLC (cohort 2).

3. Participants who have previously received the following treatments:

Cohort 1 (Melanoma): Participants who experienced documented radiographic disease progression during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibody. If the tumor was BRAF V600 mutation-positive, participants also received a BRAF inhibitor with or without a MEK inhibitor.

Cohort 2 (NSCLC): Participants who had previously received no more than 3 lines of prior therapy, and

● Participants without oncogene-driven tumors who experienced documented progression of radiation-related disease during systemic therapy with PD-1/PD-L1 blocking antibodies or within 12 weeks of the last dose of PD-1/PD-L1 blocking antibodies.

● Or for participants with tumors driven by oncogenes for which effective targeted therapies are available (e.g., EGFR, anaplastic lymphoma kinase [ALK], ROS proto-oncogene [ROS], or proto-oncogenes encoding MET tyrosine receptor kinase [MET]), who have experienced documented radiological disease progression during or after at least one line of appropriate targeted therapy and platinum-based dual chemotherapy, or during systemic therapy with PD-1/PD-L1 blocking antibodies, or within 12 weeks after the last dose of a PD-1/PD-L1 blocking antibody.

4. Participants whose ECOG fitness level is 0 or 1.

5. Participants assessed as having at least one resectable lesion

6. Previously irradiated lesions must have radiographic progression before collection.

7. Participants who are of childbearing age or whose partners are of childbearing age must be willing to use an approved, highly effective method of contraception during the treatment period and for 12 months after receiving all protocol-related treatments.

Key Exclusion Criteria

Participants were excluded from the study if they met any of the following criteria:

1. Participants with severe heart disease.

2. Participants with uveal/ocular melanoma.

3. Participants with symptomatic and untreated brain metastases.

4. Participants with any form of primary immunodeficiency.

5. Participants who have had another primary malignant disease within the previous 3 years.

6. Participants who have received or will receive a live or attenuated vaccine within 28 days prior to the start of NMA-LD.

References for Example 22

Besser MJ, Shapira-Frommer R, ltzhaki O, et al. Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma: Intent-to-Treat Analysis and Efficacy after Failure to Prior Immunotherapies. ClinCancer Res. 2013; 19(17): 4792-800.

Creelan BC, Wang C, Teer J, et al. Tumor-Infiltrating LymphocyteTreatment for Anti-PD-1-Resistant Metastatic Lung Cancer: A Phase 1 Trial. NatMed. 2021; 27(8): 1410-1418.

Howlader N, Noone A, Krapcho M, et al. 2020. SEER Cancer Statistics Review, 1975-2017. National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2017/, based on November 2019 SEER data submission.

Larkin JMG, Sarnaik A, Chesney JA, et al. Lifileucel(LN-144), aCryopreserved Autologous Tumor Infiltrating Lymphocyte(TIL)Ther apy inPatients with Advanced Melanoma: Evaluation of Impact of Prior Anti-PD-1Therapy.J Clin Oncol.2021;39(15_suppl):9505.

Mullinax JE, Hall M, Prabhakaran S, et al. Combination of lpilimumab and Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes for Patients withMetastatic Melanoma. Front Oncol. 2018; 8:44.

O'Malley D, Lee SM, Psyrri A, et al.Phase 2Efficacy and Safety ofAutologous Tumor-Infiltrating Lymphocyte(TIL)Cell Therapy in Combinati on with Pembrolizumab in Immune Checkpoint Inhibitor-Patients with Advanced Cancers J Immunother Cancer. 2021; 9(suppl 2, abs492): A523-4.

Rosenberg SA, Yang JC, Sherry RM, et al. Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell TransferImmunotherapy. Clin Cancer Res. 2011;17(13):4550-7.

Sarnaik AA, Hamid O, Khushalani NI, et al. Lifileucel, a Tumor-Infiltrating Lymphocyte Therapy, in Metastatic Melanoma. J Clin Oncol. 2021: JCO2100612.

Schoenfeld AJ, Lee SM, Paz-Ares L, et al. First Phase 2Results of Autologous Tumor-Infiltrating Lymphocyte (TIL; LN-145) Monotherapy in Patient swith Advanced, Immune Checkpoint Inhibitor-Treated, Non-Small Cell Lung Cancer (NsCLC). J Immunother Cancer. 2021; 9 (suppl2, abs 458): A486-7.

Steininger J, Gellrich FF, Schulz A, et al. Systemic Therapy of Metastatic Melanoma: On the Road to 75Cure. Cancers (Basel). 2021; 13(6).

Example 23: Evaluation of Xenon Electroporation Instrument

A study was conducted to determine whether a thermoexcimer could be used in the process of generating PD-1KO TILs. The results are summarized in Figure 76.

Method Overview

Activated or pre-REP TILs are used for experiments.

●REP TIL: Demonstration Day, Neon Experiment 1, Xenon Experiment 1

●Pre-REP TIL: Xenon Experiment 3, Xenon Experiment 4

●Activate for 5 days using TransAct

Cell preparation days for electroporation (same across experiments):

● Equilibrate the buffer solution to room temperature.

● Wash TIL with 1x PBS

● Wash TIL with 1x electroporation buffer (Thermo-CTS ^™ Xenon ^™ Genome Editing Buffer)

● Centrifuge and resuspend in the final volume of electroporation buffer.

○ A single injection requires an exact volume of 1 ml per sample (maximum 100e6/ml).

○ The neon volume can be 10ul or 100ul (maximum 20e6/ml).

●Add mRNA (GFP 1ug/1e6 cells, TALEN 4ug/1e6 cells)

●Electroplution

● Rest overnight in CM2 + 3000 IU/ml IL-2 in G-Rex10 (if using >10e6, split into 2 G-Rex10s).

●37℃, for GFP mRNA

●30℃, for TALEN mRNA

● Collect resting samples and bacterial strains using a 150µm cell filter.

● Perform cell counting and flow staining or REP (Xenon Experiment 4).

Experiment 1: Demonstration Day, Thermo

● Activated REP TIL (L4346) was tested using GFP mRNA.

● Thawing (Day 0) and Activation (Day 1) 50e6+TransAct

● 1ug/1e GFP mRNA per 6 cells (stock solution, 1mg/ml)

● Electroporation using Thermo's recommended optimization scheme

● Single injection (range: 2e7-1e8 cells per 1ml) – 20e6/ml

●Neon – 2e6/100ul

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 77, the results indicate that after 24 hours, 1700/20/1 and 2300/3/4 showed higher GFP expression and recovery rates.

Experiment 2: Neon Experiment 1

●Objective: To optimize two electroporation setups identified during the xenon demonstration using neon and compare them with BTX.

●Cell concentration: 2e6 cells per 100 μl of neon-1

● Xenon backbone electroporation setup:

○1700/20/1

○2300/3/4

● Change the duration (ms) and number of pulses while keeping the voltage setting constant.

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

○ 24 hours later, Annexin V was detected by flow cytometry.

As shown in Figure 78, the results indicate that the 2300/3/4 Thermo recommended setting (5*) has the highest GFP expression and 60% recovery rate, and the recovery rate is improved at the 2300V voltage setting with shorter duration (2ms) and fewer pulses (3), and GFP expression is reduced.

Experiment 3: Xenon Experiment 1

●Objective: To determine the effect of cell concentration on xenon-GFP expression and recovery rate.

●N = 1 activated REP TIL sample (EP11231)

●Xenon and BTX

●Electroporation conditions: 2300/3/4, selected based on maximum GFP expression and 60% recovery rate.

●Use a single injection to test cell concentration

○1e6/ml

○5e6/ml

○10e6/ml

○25e6/ml

●BTX Comparison

○10e6+GFP (in 400 μl, therefore the concentration is 25e6/ml)

○2e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 79, the results indicate that GFP expression was slightly reduced when xenon was used compared to BTX. GFP expression was not affected by cell concentration, but the recovery rate decreased and apoptosis increased at lower cell concentrations. After 24 hours, 2300/3/4 appeared to be toxic at low cell concentrations.

Experiment 4: Xenon Experiment 3

Objective: To determine the effect of cell concentration (low/high) on xenon GFP expression and recovery rate using the EP setting that exhibits the highest recovery rate.

●N = 1 activated pre-REP TIL sample (K7136)

●Xenon and BTX

●Electroplution conditions:

○1400/30/1

○1700/20/1

○2300/2/3

○2500/2/5

●Use a single injection to test cell concentration

○5e6/ml

○25e6/ml

●BTX Comparison

○5e6+GFP (in 200 μl, therefore the concentration is 25e6/ml)

○1e6 Simulation

●Test parameters:

○Resuscitation rate and survival rate

○ 24 hours later, GFP (L/D aqua and FITC, for GFP) was measured by flow cytometry.

As shown in Figure 80, the results indicate that high GFP expression exists between samples, comparable to that of BTX, and the difference between 5e6 and 25e6 is minimal. For EP settings 1400/30/1 and 2300/2/3, there are similar recovery rates between 5e6 and 25e6, and after 24 hours, the recovery rate of 2300/2/3 is comparable to that of BTX.

Experiment 5: Xenon Experiment 4

●Objective: To compare the Xenon electroporator and the BTX electroporator using TALEN mRNA.

●N = 1 activated pre-REP TIL sample (L4374)

●Xenon and BTX

●Electroporation conditions:

○2300/2/3

●Conditions:

Xenon (Talen(25e6))

Xenon simulant (5e6)

○BTX Talen (25e6)

○BTX emulator (5e6)

●Test parameters:

○Resuscitation rate and survival rate

○ PD-1 KO based on flow cytometry

■ PD-1 phase KO flow cytometry data operated on resting samples after electroporation (without REP)

■REP collects final PD-1KO flow cytometry data from samples

As shown in Figure 81, the results showed that the recovery rates of xenon and BTX were similar between 5e6 and 25e6, with xenon having a slightly higher recovery rate. The PD-1 KO efficiency (resting cells after electroporation) was similar between xenon (89%) and BTX (86%). Furthermore, for the analysis of PD-1 expression and KO efficiency in REP samples, the KO efficiency of xenon (83%) was slightly higher than that of BTX (76%).

in conclusion

The study identified an electroporation setting of 2300/2/3 for xenon that resulted in high cell resuscitation rates (comparable to BTX) at both low and high cell concentrations, high GFP expression (comparable to BTX) at both low and high cell concentrations, and high PD-1 KO efficiency (comparable to BTX).

The above embodiments are provided to give those skilled in the art a complete disclosure and description of how to prepare and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications to the above-described patterns of the invention that are obvious to those skilled in the art are intended to fall within the scope of the following claims. All patents and disclosures mentioned in this specification indicate the skill level of those skilled in the art to which this invention pertains.

All headings and section titles are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different headings and sections as needed, in accordance with the spirit and scope of the invention described herein.

All references cited in this article are incorporated herein by reference in their entirety for all purposes, as if each individual publication or patent or patent application were specifically and individually indicated to be incorporated herein by reference in their entirety for all purposes.

As will be apparent to those skilled in the art, various modifications and alterations can be made thereto without departing from the spirit and scope of this application. The specific embodiments and examples described herein are provided by way of example only, and this application is limited only to the appended claims and the full scope of their equivalents.

Claims (223)

1. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population;

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(e) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

2. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from the subject or patient is cultured in a first cell culture medium containing IL-2 for approximately 3–9 days to generate a second population of TILs;

(b) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(c) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population; and

(d) The fourth TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

3. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) The fourth TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

4. A method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising the following steps:

(a) Obtaining and/or receiving the first TIL population from tumor tissue samples produced by surgical resection, puncture biopsy, core needle biopsy, mini biopsy or other means of obtaining tumor tissue from a patient or subject;

(b) The tumor tissue is added to a closed system and a first expansion is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first breathable surface area, and wherein the first expansion is performed for about 3-9 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–7 days using anti-CD3 agonist beads or antibodies, or CD3 agonist and CD28 agonist beads or antibodies, to generate a third TIL population;

(d) Gene editing of at least a portion of the third TIL population to generate a fourth TIL population;

(e) A second expansion is performed by culturing the fourth TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fifth TIL population, wherein the second expansion is carried out for approximately 5-15 days to obtain the fifth TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fifth TIL population is a therapeutic TIL population; and

(f) Collect the therapeutic TIL population obtained from step (e), wherein each of steps (b) to (f) is performed in a closed, sterile system, and wherein the transition from step (b) to step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), and/or the transition from step (e) to step (f) is performed without opening the system.

5. The method according to any one of claims 1-4, further comprising:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

6. The method of claim 5, wherein the enzyme medium comprises a DNA enzyme.

7. The method of claim 5 or 6, wherein the enzyme medium comprises collagenase.

8. The method of any one of claims 5-7, wherein the enzyme medium comprises a neutral protease.

9. The method of any one of claims 5-8, wherein the enzyme medium comprises hyaluronidase.

10. The method of any one of claims 1-9, wherein the step of culturing or rapidly expanding the fourth TIL population is performed by: culturing the fourth TIL population in a second cell culture medium for a first period of about 1-7 days, at the end of the first period the fourth TIL population is split into multiple subcultures, each of the subcultures is cultured in a third cell culture medium containing IL-2 for a second period of about 3-7 days, and at the end of the second period the subcultures are combined to provide an expanded number of TILs or the therapeutic TIL population.

11. The method of claim 10, wherein the first culture period is approximately 5 days.

12. The method of claim 10 or 11, wherein the second culture period is approximately 4 days.

13. The method of claim 10 or 11, wherein the second culture period is approximately 5 days.

14. The method of any one of claims 1-13, wherein the step of activating the second TIL population is performed using anti-CD3 agonist beads or antibodies.

15. The method of claim 14, wherein the step of activating the second TIL population is performed using OKT-3.

16. The method of claim 15, wherein the step of activating the second TIL population is performed using 300 ng/mL of OKT-3.

17. The method of any one of claims 1-16, wherein the step of activating the second TIL population is performed using anti-CD3 agonist and anti-CD28 agonist beads or antibodies.

18. The method of claim 17, wherein the step of activating the second TIL population is performed using TransAct.

19. The method of claim 18, wherein the step of activating the second TIL population is performed using TransAct diluted at a ratio of 1:10, 1:17.5, or 1:100.

20. The method of any one of claims 1-19, wherein the step of activating the second TIL population is performed for approximately 2 days.

21. The method of any one of claims 1-19, wherein the step of activating the second TIL population is performed for approximately 3 days.

22. The method of any one of claims 1-19, wherein the step of activating the second TIL population is performed for approximately 4 days.

23. The method of any one of claims 1-19, wherein the step of activating the second TIL population is performed for approximately 5 days.

24. The method of any one of claims 1-23, wherein the step of culturing the first TIL population is performed for approximately 3 days.

25. The method of any one of claims 1-23, wherein the step of culturing the first TIL population is performed for approximately 5 days.

26. The method of any one of claims 1-23, wherein the step of culturing the first TIL population is performed for approximately 7 days.

27. The method of any one of claims 1-26, wherein the step of culturing the fourth TIL population is performed for approximately 8 days.

28. The method of any one of claims 1-26, wherein the step of culturing the fourth TIL population is performed for approximately 9 days.

29. The method of any one of claims 1-26, wherein the step of culturing the fourth TIL population is performed for about 8-9 days.

30. The method of any one of claims 1-26, wherein the step of culturing the fourth TIL population is performed for approximately 10 days.

31. The method of any one of claims 1-26, wherein the step of culturing the fourth TIL population is performed for about 8-10 days.

32. The method of any one of claims 1-31, wherein all steps are completed within a period of approximately 22 days.

33. The method of any one of claims 1-31, wherein all steps are completed within a period of approximately 19-22 days.

34. The method of any one of claims 1-31, wherein all steps are completed within a period of about 19-20 days.

35. The method of any one of claims 1-31, wherein all steps are completed within a period of about 20-22 days.

36. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) The first TIL population was cultured in a first cell culture medium containing IL-2 and OKT-3 for about 3-9 days to produce a second TIL population;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

37. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first population of TILs obtained and/or received from tumor tissue excised from a subject or patient is cultured in a first cell culture medium containing IL-2 and OKT-3 for approximately 3–9 days to generate a second population of TILs;

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

38. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Obtaining and/or receiving the first TIL population from tumor tissue excised from the subject or patient;

(b) Initial expansion (or initiation of first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APC), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days;

(c) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(d) The third TIL population is rapidly amplified in a second cell culture medium to obtain an amplified number of TILs; wherein the second cell culture medium contains IL-2, OKT-3 and APC; and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

39. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) Initial expansion (or initiation of first expansion) of a first population of TILs obtained and/or received from tumor tissue excised from a subject or patient in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2, optionally OKT-3 and optionally antigen-presenting cells (APCs), wherein the initiation of first expansion is carried out over a period of approximately 3 to 8 days.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population is subjected to rapid second amplification in a second cell culture medium containing IL-2, OKT-3 and APC; and the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification.

40. The method of any one of claims 36-39, further comprising:

The tumor tissue is digested in an enzyme medium to produce tumor digestate.

41. The method of claim 40, wherein the enzyme medium comprises a DNA enzyme.

42. The method of claim 40 or 41, wherein the enzyme medium comprises collagenase.

43. The method of any one of claims 40-42, wherein the enzyme medium comprises a neutral protease.

44. The method of any one of claims 40-43, wherein the enzyme medium comprises hyaluronidase.

45. A method for preparing expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) A first TIL population obtained by digesting tumor tissue excised from a subject or patient in an enzyme medium in a first cell culture medium containing IL-2 and OKT-3 for about 3–9 days to produce a second TIL population.

(b) Gene editing of at least a portion of the second TIL population to generate a third TIL population; and

(c) The third TIL population was cultured for approximately 5–15 days in a second cell culture medium containing antigen-presenting cells (APCs), OKT-3, and IL-2 to produce an expanded number of TILs.

46. The method of any one of claims 36-45, wherein the step of culturing or initially expanding the first TIL population comprises culturing the first TIL population in the first cell culture medium containing IL-2 for about 3 days, followed by culturing the first TIL population in the cell culture medium containing IL-2 and OKT-3 for 2-6 days.

47. The method of any one of claims 36-46, wherein the step of culturing or rapidly amplifying the third TIL population is performed by: culturing the third TIL population in a second cell culture medium for a first period of about 1-7 days, at the end of the first period the third TIL population is split into multiple subcultures, each subculture is cultured in a third cell culture medium containing IL-2 for a second period of about 3-7 days, and at the end of the second period the subcultures are combined to provide an amplified number of TILs.

48. The method of claim 47, wherein the first culture period is about 5 days.

49. The method of claim 47 or 48, wherein the second culture period is approximately 4 days.

50. The method of claim 47 or 48, wherein the second culture period is approximately 5 days.

51. The method of any one of claims 36-50, wherein the step of culturing the first TIL population is performed for approximately 3 days.

52. The method of any one of claims 36-50, wherein the step of culturing the first TIL population is performed for approximately 5 days.

53. The method of any one of claims 36-50, wherein the step of culturing the first TIL population is performed for approximately 7 days.

54. The method of any one of claims 36-53, wherein the step of culturing the third TIL population is performed for approximately 8 days.

55. The method of any one of claims 36-53, wherein the step of culturing the third TIL population is performed for approximately 9 days.

56. The method of any one of claims 36-53, wherein the step of culturing the third TIL population is performed for approximately 8-9 days.

57. The method of any one of claims 36-53, wherein the step of culturing the third TIL population is performed for approximately 10 days.

58. The method of any one of claims 36-53, wherein the step of culturing the third TIL population is performed for about 8-10 days.

59. The method of any one of claims 36-58, wherein all steps are completed within a period of approximately 22 days.

60. The method of any one of claims 36-58, wherein all steps are completed within a period of about 20 days.

61. The method of any one of claims 36-58, wherein all steps are completed within a period of approximately 22 days.

62. The method of any one of claims 36-58, wherein all steps are completed within a period of approximately 19-22 days.

63. The method of any one of claims 36-58, wherein all steps are completed within a period of about 19-20 days.

64. The method of any one of claims 36-58, wherein all steps are completed within a period of about 20-22 days.

65. The method of any one of claims 36-58, wherein all steps are completed within a period of about 16-18 days.

66. The method of any one of claims 36-65, wherein the step of culturing or initially amplifying the first TIL population in the first culture medium further comprises anti-CD3 and anti-CD28 beads or antibodies.

67. The method of claim 66, wherein the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct.

68. The method of claim 67, wherein the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

69. The method of any one of claims 36-68, wherein the first culture medium comprises 300 ng/mL of OKT-3.

70. The method of any one of claims 36-69, wherein the step of culturing or initially amplifying the first TIL population comprises culturing the first TIL population for about 3 days in a first cell culture medium containing IL-2 and anti-CD3 and anti-CD28 beads or antibodies, and then culturing the first TIL population for 2-4 days in a cell culture medium containing IL-2 and OKT-3.

71. The method of claim 70, wherein the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct.

72. The method of claim 70, wherein the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct diluted at 1:10, 1:17.5, or 1:100.

73. The method of any one of claims 70-72, wherein the first culture medium comprises 300 ng/mL of OKT-3.

74. The method of any one of claims 1-3 or 5-68, wherein the expanded number of TILs comprises a therapeutic TIL population.

75. The method of any one of claims 1-74, wherein the step of gene editing at least a portion of the second or third TIL population comprises a sterile electroporation step of the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

76. The method of any one of claims 1-74, wherein the step of gene editing at least a portion of the second or third TIL population comprises a sterile electroporation step of the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

77. The method of claim 76, wherein the electroporation step comprises a single electroporation event mediating the transfer of the at least two gene editors.

78. The method of claim 76, wherein each of the at least two gene editors in the electroporation step is transferred independently of the transfer of any other gene editor via the electroporation event.

79. The method of claim 78, wherein the electroporation step further includes a rest period following each electroporation event.

80. The method of claim 79, wherein the electroporation step comprises a first electroporation event mediating the transfer of a first gene editor for regulating the expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor for regulating the expression of a second protein, and a second resting period, wherein the first resting period is the same as or different from the second resting period.

81. The method of claim 80, wherein the first resting period and the second resting period comprise incubating the third or fourth TIL population in a second cell culture medium containing IL-2 and/or IL-15.

82. The method of claim 81, wherein the first resting period and the second resting period comprise incubating the third or fourth TIL population in a second cell culture medium containing 300 IU/mL, 1000 IU/mL or 6000 IU/mL of IL-2.

83. The method of claim 81, wherein the first resting period and the second resting period comprise incubating the third or fourth TIL population in a second cell culture medium containing 15 ng/mL IL-15.

84. The method of any one of claims 80-78, wherein the first resting period and the second resting period comprise incubating the third or fourth TIL population at about 30-40°C and about 5% _CO2 .

85. The method of claim 84, wherein the first resting period and the second resting period comprise incubating the third or fourth TIL population at about 25, 28, 30, 32, 35 or 37°C and about 5% _CO2 .

86. The method of any one of claims 80-85, wherein the first resting period and the second resting period are independently about 10 hours to 5 days.

87. The method of claim 86, wherein the first resting period and the second resting period are independently about 10 hours to 3 days.

88. The method of claim 87, wherein the first resting period is about 1 to 3 days.

89. The method of claim 87, wherein the first resting period is about 3 days.

90. The method of any one of claims 87-89, wherein the second rest period is about 10 hours to 1 day.

91. The method of any one of claims 87-89, wherein the second resting period is about 12 hours to 24 hours.

92. The method of any one of claims 87-89, wherein the second resting period is from about 15 hours to about 18 hours.

93. The method of any one of claims 87-89, wherein the second resting period comprises incubating the third or fourth TIL population at about 30°C in a cell culture medium containing IL-2 for about 15 to 23 hours.

94. The method of any one of claims 87-89, wherein the second resting period comprises incubating the third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 23 hours.

95. The method of any one of claims 87-89, wherein the second resting period comprises incubating the third or fourth TIL population at 37°C for about one hour in a cell culture medium containing IL-2, followed by incubation at about 30°C for about 15 to 22 hours.

96. The method of claim 87, wherein the first rest period is about 3 days and the second rest period is about 10 to 16 hours.

97. The method of any one of claims 75-96, wherein the second or third TIL population is washed in a cell perforation buffer prior to the electroporation step.

98. The method of claim 75 or 97, wherein the at least one gene editor is a TALE nuclease system for regulating the expression of at least one protein.

99. The method of claim 98, wherein the at least one gene editor comprises a TALE nuclease system for regulating PD-1 expression.

100. The method of claim 98, wherein the at least one gene editor comprises a TALE nuclease system that regulates CTLA-4 expression.

101. The method of claim 98, wherein the at least one gene editor comprises a TALE nuclease system that regulates LAG-3 expression.

102. The method of claim 98, wherein the at least one gene editor comprises a TALE nuclease system that regulates CISH expression.

103. The method of claim 98, wherein the at least one gene editor comprises a TALE nuclease system for regulating CBL-B expression.

104. The method of claim 98, wherein the at least one gene editor comprises a TALE nuclease system for regulating TIGIT expression.

105. The method of any one of claims 76-97, wherein the at least two gene editors comprise a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

106. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT and/or CBL-B.

107. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CTLA-4.

108. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and LAG-3.

109. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CISH.

110. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CBL-B.

111. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and TIGIT.

112. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and LAG-3.

113. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CISH.

114. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CBL-B.

115. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CISH.

116. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CBL-B.

117. The method of claim 105, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CISH and CBL-B.

118. The method of claim 105, wherein the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

119. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

120. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

121. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

122. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

123. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

124. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

125. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

126. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

127. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

128. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

129. The method of claim 118, wherein the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

130. The method of claim 118, wherein the first protein is PD-1 and the second protein is CTLA-4.

131. The method of claim 118, wherein the first protein is CTLA-4 and the second protein is PD-1.

132. The method of claim 118, wherein the first protein is PD-1 and the second protein is LAG-3.

133. The method of claim 118, wherein the first protein is LAG-3 and the second protein is PD-1.

134. The method of claim 118, wherein the first protein is PD-1 and the second protein is CISH.

135. The method of claim 118, wherein the first protein is CISH and the second protein is PD-1.

136. The method of claim 118, wherein the first protein is PD-1 and the second protein is CBL-B.

137. The method of claim 118, wherein the first protein is CBL-B and the second protein is PD-1.

138. The method of claim 118, wherein the first protein is PD-1 and the second protein is TIGIT.

139. The method of claim 118, wherein the first protein is TIGIT and the second protein is PD-1.

140. The method of claim 118, wherein the first protein is CTLA-4 and the second protein is LAG-3.

141. The method of claim 118, wherein the first protein is LAG-3 and the second protein is CTLA-4.

142. The method of claim 118, wherein the first protein is CTLA-4 and the second protein is CISH.

143. The method of claim 118, wherein the first protein is CISH and the second protein is CTLA-4.

144. The method of claim 118, wherein the first protein is CTLA-4 and the second protein is CBL-B.

145. The method of claim 118, wherein the first protein is CBL-B and the second protein is CTLA-4.

146. The method of claim 118, wherein the first protein is LAG-3 and the second protein is CISH.

147. The method of claim 118, wherein the first protein is CISH and the second protein is LAG-3.

148. The method of claim 118, wherein the first protein is LAG-3 and the second protein is CBL-B.

149. The method of claim 118, wherein the first protein is CBL-B and the second protein is LAG-3.

150. The method of claim 118, wherein the first protein is CISH and the second protein is CBL-B.

151. The method of claim 118, wherein the first protein is CBL-B and the second protein is CISH.

152. The method of claim 118, wherein the first protein or the second protein is PD-1.

153. The method of claim 118, wherein the first protein or the second protein is CTLA-4.

154. The method of claim 118, wherein the first protein or the second protein is LAG-3.

155. The method of claim 118, wherein the first protein or the second protein is CISH.

156. The method of claim 118, wherein the first protein or the second protein is CBL-B.

157. The method of claim 118, wherein the first protein or the second protein is TIGIT.

158. The method of any one of claims 76-97 or 105-157, wherein the first gene editor downregulates the expression of the first protein and the second gene editor downregulates the expression of the second protein.

159. The method of any one of claims 1-158, wherein the antigen-presenting cell (APC) is a PBMC.

160. The method of claim 159, wherein the PBMC is irradiated and allogeneic.

161. The method according to any one of claims 1-158, wherein the antigen-presenting cell is an artificial antigen-presenting cell.

162. The method according to any one of claims 1-161, wherein the IL-2 concentration is from about 10,000 IU/mL to about 5,000 IU/mL.

163. The method according to any one of claims 1-162, wherein the first cell culture medium and/or the second cell culture medium further comprises a 4-1BB agonist and/or an OX40 agonist.

164. The method according to any one of claims 1-163, wherein the tumor tissue is processed into a plurality of tumor fragments.

165. The method of claim 164, wherein the tumor fragment is added to the closed system.

166. The method of claim 165, wherein 150 or fewer of the segments, 100 or fewer of the segments, or 50 or fewer of the segments are added to the closed system.

167. A gene-edited tumor-infiltrating lymphocyte (TIL) population, said TIL population comprising an expanded TIL population, wherein the expression of at least one protein is regulated by a gene editor transferred to at least a portion of said expanded TIL population.

168. The gene-edited TIL population of claim 167, wherein the gene editor is a TALE nuclease system for regulating the expression of the at least one protein.

169. The gene-edited TIL population of claim 168, wherein the at least one protein is PD-1.

170. The gene-edited TIL population of claim 168, wherein the at least one protein is CTLA-4.

171. The gene-edited TIL population of claim 168, wherein the at least one protein is LAG-3.

172. The gene-edited TIL population of claim 168, wherein the at least one protein is CISH.

173. The gene-edited TIL population of claim 168, wherein the at least one protein is CBL-B.

174. The gene-edited TIL population of claim 168, wherein the at least one protein is TIGIT.

175. The gene-edited TIL population of claim 167, wherein the expression of at least two proteins is regulated by at least two gene editors transferred to at least a portion of the amplified TIL population, wherein the at least two gene editors include a first gene editor comprising a first TALE nuclease system for regulating the expression of a first protein and a second gene editor comprising a second TALE nuclease system for regulating the expression of a second protein.

176. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT and CBL-B, provided that the first protein is different from the second protein.

177. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

178. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

179. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

180. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

181. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

182. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

183. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

184. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

185. The gene-edited TIL population of claim 175, wherein the first TALE protein and the second TALE protein are selected from the group consisting of LAG-3 and CISH.

186. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

187. The gene-edited TIL population of claim 175, wherein the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

188. The gene-edited TIL population as described in any one of claims 167-187, wherein the TIL population is produced by the method described in any one of claims 1-166.

189. A pharmaceutical composition comprising the gene-edited TIL population of any one of claims 167-188 and a pharmaceutically acceptable vector.

190. A method for treating a subject with cancer, the method comprising administering a therapeutically effective dose of the gene-edited TIL population of any one of claims 167-188 or the pharmaceutical composition of claim 189.

191. The method of claim 190, wherein the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

192. A method for treating a subject with cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), comprising:

(a) Obtaining a first TIL population from a tumor excised from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) The tumor fragment is added to a closed system and a first amplification is performed by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first amplification is performed in a closed container providing a first breathable surface area, wherein the first amplification is performed for about 3-8 days to obtain the second TIL population.

(c) Activate the second TIL population for 1–6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to generate the third TIL population;

(e) Performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor;

(f) Allow the third TIL population to rest for approximately one day;

(g) A second expansion is performed by culturing the third TIL population in a second cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a fourth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the third TIL population, wherein the second expansion is performed in a closed container providing a second breathable surface area, and wherein the fourth TIL population is a therapeutic TIL population.

(h) Collect the therapeutic TIL population obtained from step (e) to provide the collected TIL population, wherein one or more of steps (a) to (h) are performed in a closed, sterile system;

(i) The collected TIL population is transferred to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system;

(j) The collected TIL population was cryopreserved using a dimethyl sulfoxide-based cryopreservation medium; and

(k) Administer a therapeutically effective dose of the collected TIL population from the infusion bag to the patient;

The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and/or CBL-B.

193. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting PD-1 expression.

194. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CTLA-4 expression.

195. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting LAG-3 expression.

196. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CISH expression.

197. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CBL-B expression.

198. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting TIGIT expression.

199. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting PD-1 and CTLA-4 expression.

200. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting PD-1 and LAG-3 expression.

201. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting PD-1 and CISH expression.

202. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting PD-1 and CBL-B expression.

203. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting PD-1 and TIGIT expression.

204. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CTLA-4 and LAG-3 expression.

205. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CTLA-4 and CISH expression.

206. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CTLA-4 and CBL-B expression.

207. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CTLA-4 and TIGIT expression.

208. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting LAG-3 and CISH expression.

209. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting LAG-3 and CBL-B expression.

210. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting LAG-3 and TIGIT expression.

211. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CISH and CBL-B expression.

212. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CISH and TIGIT expression.

213. The method of claim 192, wherein the electroporation step comprises delivering a TALEN system for inhibiting CBL-B and TIGIT expression.

214. The method of any one of claims 192-213, wherein the therapeutically effective dose of TIL is about 1 × ^10⁹ to about 1 × ^10¹¹ TILs.

215. The method of any one of claims 192-214, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to administering a therapeutically effective dose of the collected TIL population in step (k).

216. The method of any one of claims 192-215, further comprising the step of treating the patient with a high-dose IL-2 regimen initiated the day after the patient has been given the therapeutically effective dose of the collected TIL population in step (k).

217. The method of any one of claims 192-216, wherein the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

218. The method of any one of claims 192-216, wherein the cancer is melanoma.

219. The method of claim 218, wherein the cancer is metastatic melanoma.

220. The method of any one of claims 192-216, wherein the cancer is NSCLC.

221. The method of claim 220, wherein the cancer is metastatic NSCLC.

222. The method of any one of claims 192-221, wherein the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

223. Use of a composition, product, process, or system comprising TIL produced by any of the methods described herein, said use including the use of TIL in the preparation of a medicament for treating a subject suffering from cancer, characterized by one or more elements disclosed in this application.