CN117940557A

CN117940557A - Method for preparing modified tumor-infiltrating lymphocytes and application of modified tumor-infiltrating lymphocytes in adoptive cell therapy

Info

Publication number: CN117940557A
Application number: CN202280026986.2A
Authority: CN
Inventors: 张永亮; R·库巴斯; F·G·福格特; M·法尔迪; C·沙尔蒂尔库托
Original assignee: Iovance Biotherapeutics Inc
Current assignee: Iovance Biotherapeutics Inc
Priority date: 2021-01-29
Filing date: 2022-01-28
Publication date: 2024-04-26

Abstract

Provided herein are compositions and methods for treating cancer using a modified TIL, wherein the modified TIL comprises one or more immunomodulatory agents (e.g., cytokines) associated with its cell surface. Immunomodulators associated with TIL provide local immunostimulatory effects, which may advantageously enhance TIL survival, proliferation, and/or anti-tumor activity in patient recipients. Accordingly, the compositions and methods disclosed herein provide effective cancer therapies.

Description

Method for preparing modified tumor-infiltrating lymphocytes and application of modified tumor-infiltrating lymphocytes in adoptive cell therapy

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/143,736, U.S. provisional application No. 63/146,486, U.S. provisional application No. 63/224,360, U.S. provisional application No. 63/277,571, and U.S. provisional application No. 63/285,956, U.S. provisional application No. 63/277,571, and U.S. provisional application No. 63/285,956, U.S. provisional application No. 2021, 11, and 3, U.S. provisional application No. 2021, 12, and 3, all of which are incorporated herein by reference in their entirety.

Background

Adoptive cell therapy with TIL cultured ex vivo by Rapid Expansion Protocol (REP) has resulted in successful adoptive cell therapy following host immunosuppression in patients with cancer. However, in some cases, the survival and anti-tumor activity of the metastasized TIL may decrease after metastasis to the patient.

Administration of supportive immunostimulants (e.g., cytokines) has been investigated to enhance T cell therapies. However, such immunostimulants require high systemic doses, which can cause undesirable toxicity.

Thus, there remains a need for improved TIL therapies for the treatment of cancer.

Disclosure of Invention

In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), optionally wherein the patient or subject has received at least one prior therapy, a portion of the TILs being modified TILs such that each of the modified TILs comprises an immunomodulatory composition associated with a surface membrane thereof.

In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Obtaining and/or receiving a first TIL population from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) Adding a first TIL population to a closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area, the first amplification being performed for about 3 days to 14 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(d) Performing a second amplification by supplementing a cell culture medium of a second population of TILs with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a third population of TILs, wherein the second amplification is performed for about 7 days to 14 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(e) Collecting the population of therapeutic TILs obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system;

(f) Transferring the collected TIL population from step (e) to an infusion bag, wherein the transferring of steps (e) to (f) is performed without opening the system;

(g) Cryopreserving the infusion bag from step (f) containing the collected TIL population by a cryopreservation process;

(h) Administering to the subject a therapeutically effective dose of a third population of TILs from the infusion bag of step (g); and

(I) Modifying a portion of the TILs at any time prior to administration of (h) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) Adding tumor fragments into the closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2, producing a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area, the first amplification being performed for about 3 days to 11 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(d) Performing a second amplification by supplementing a cell culture medium of a second population of TILs with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a third population of TILs, wherein the second amplification is performed for about 7 days to 11 days to obtain the third population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(e) Collecting a third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system;

(f) Transferring the collected third TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) is performed without opening the system;

In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Obtaining and/or receiving a first population of TILs from surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a patient or subject's cancer,

(B) Adding a first TIL population to a closed system;

In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Resecting a tumor from a subject or patient, the tumor comprising a first population of TILs, optionally by surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer;

(b) Treating the tumor into a plurality of tumor fragments and adding the tumor fragments to the closed system;

(h) Administering a therapeutically effective dose of the third TIL population from the infusion bag of step (g) to a subject or patient having cancer; and

In other aspects, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Obtaining and/or receiving a first population of TILs from a surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a subject or patient;

(c) Contacting a first population of TILs with a first cell culture medium;

(d) Performing an initial expansion of the first population of TILs (or initiating a first expansion (PRIMING FIRST expansion)) in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD 3 antibody), and optionally Antigen Presenting Cells (APC), initiating the first expansion for a period of 1 to 8 days;

(e) Performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD 3 antibody) and APC; the rapid amplification is performed for a period of time less than 14 days, alternatively, the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the start of the rapid second amplification;

(f) Collecting a third TIL population;

(g) Administering a therapeutically effective portion of a third population of TILs to a subject or patient having cancer; and

(H) Modifying a portion of the TILs at any time prior to administration of (g) such that each of the modified TILs comprises an immunomodulatory composition bound to its surface membrane.

In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Resecting a tumor from a cancer of a subject or patient, the tumor comprising a first population of TILs, optionally by surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample from the cancer that contains a mixture of tumor and TIL cells;

(b) Breaking up the tumor into tumor fragments;

(c) Contacting tumor fragments with a first cell culture medium;

(d) Performing an initial expansion of the first population of TILs (or initiating the first expansion) in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD 3 antibody), and optionally Antigen Presenting Cells (APC), wherein initiating the first expansion is performed for a period of 1 to 8 days;

(f) Collecting a third TIL population;

(I) Modifying a portion of the TILs at any time prior to administration (g) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

In one aspect, provided herein is a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, comprising:

(a) Obtaining and/or receiving a first TIL population of tumors from a cancer resection from a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments;

(b) Selecting PD-l positive TIL from the first TIL population in step (a), obtaining a PD-l enriched TIL population;

(c) Initiating a first expansion by culturing a PD-l enriched population of TILs in a first cell culture medium comprising IL-2, OKT-3 and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed in a vessel having a first gas permeable surface area, the initiating the first expansion being performed for a first period of time of about 1 to 7 days or about 1 to 8 days, obtaining a second population of TILs, the number of the second population of TILs being greater than the first population of TILs;

(d) Performing a rapid second amplification by culturing a second population of TILs in a second medium comprising IL-2, OKT-3 and APCs, resulting in a population of therapeutic TILs, wherein the number of APCs added in the rapid second amplification is at least twice the number of APCs added in step (b), the rapid second amplification being performed for a second period of about 1 to 11 days to obtain a population of therapeutic TILs, the third population of TILs being the population of therapeutic TILs, the rapid second amplification being performed in a container having a second gas permeable surface area;

(e) Collecting the therapeutic TIL population obtained from step (d);

(f) Transferring the collected TIL population from step (e) to an infusion bag, and

(G) A portion of the TILs are modified at any time during the method such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

Provided herein are methods of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(a) Obtaining and or receiving a first population of TILs from a tumor resected from a cancer of a subject or patient by processing a tumor sample obtained from the tumor into a plurality of tumor fragments;

(b) Adding a first TIL population to a closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2, producing a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area, the first amplification being performed for about 3 days to 14 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(f) Transferring the collected TIL population from step (e) to an infusion bag, wherein the transferring of steps (e) to (f) is performed without opening the system; and

(G) Modifying a portion of the TILs at any time prior to transfer to the infusion bag in step (f) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

(a) Obtaining a first population of TILs from a tumor resected from a cancer of a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments;

(b) Adding tumor fragments into the closed system;

(f) Transferring the collected third TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to step (f) is performed without opening the system; and

In another aspect, provided herein is a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(a) Obtaining and/or receiving a first population of TILs from surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer of a patient or subject,

(B) Adding a first TIL population to a closed system;

(d) Performing a second amplification by supplementing a cell culture medium of a second population of TILs with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a third population of TILs, the second amplification being performed for about 7 to 11 days to obtain the third population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(G) Modifying a portion of the TILs at any time prior to the transferring in step (f) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

In one aspect, provided herein is a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(b) Adding tumor fragments into the closed system;

In some embodiments of the methods provided herein, the first amplification is divided into a first step and a second step, wherein the method further comprises the first step of performing the first amplification by culturing the first population of TILs in a cell culture medium containing IL-2, producing TILs that are shed from tumor fragments or samples, separating TILs remaining in the tumor fragments or samples from TILs shed from the tumor fragments or samples, optionally, digesting the tumor fragments or samples to produce tumor digests, and the second step of performing the first amplification by culturing the remaining TILs in the tumor fragments or samples or tumor digests in the cell culture medium, producing the second population of TILs.

In one aspect, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(a) Obtaining and/or receiving a first population of TILs from surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer of a subject or patient;

(b) Contacting a first population of TILs with a first cell culture medium;

(c) Performing an initial expansion of the first population of TILs (or initiating the first expansion) in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD 3 antibody), and optionally Antigen Presenting Cells (APC), initiating the first expansion for a period of 1 to 8 days;

(d) Performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD 3 antibody) and APC, and the rapid amplification is performed for a period of time of 14 days or less, alternatively, the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification;

(e) Collecting a third TIL population; and

(F) Modifying a portion of the TILs at any time prior to collection in step (f) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

(a) Resecting a tumor from a cancer of a subject or patient, the tumor comprising a first population of TILs, optionally by surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a tumor sample comprising a mixture of tumor and TIL cells;

(b) Breaking up the tumor into tumor fragments;

(c) Contacting tumor fragments with a first cell culture medium;

(d) Performing an initial expansion of the first population of TILs (or initiating the first expansion) in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD 3 antibody), and optionally Antigen Presenting Cells (APC), initiating the first expansion for a period of 1 to 8 days;

(e) Performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD 3 antibody) and APC, and the rapid amplification is performed for a period of time of 14 days or less, alternatively, the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the rapid second amplification;

(f) Collecting a third TIL population; and

(G) Modifying a portion of the TILs at any time prior to collection in step (f) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

(a) Obtaining and/or receiving a first TIL population from a tumor resected from a cancer of a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments;

(b) Initiating a first expansion by culturing a first population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, and optionally Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed for a first period of about 1 to 7 days or about 1 to 8 days, obtaining a second population of TILs, the number of second population of TILs being greater than the first population of TILs;

(c) Performing a rapid second amplification by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3 and APC, producing a third population of TILs, wherein the rapid second amplification is performed for a second period of time of about 1 to 11 days, obtaining the third population of TILs, the third population of TILs being a therapeutic population of TILs;

(d) Collecting the therapeutic TIL population obtained from step (c); and

(E) Modifying a portion of the TILs at any time before or after collection in step (d) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

In some embodiments of this method, the cell culture medium in step (b) further comprises Antigen Presenting Cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

(a) Initiating a first expansion by culturing a first population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, and optionally Antigen Presenting Cells (APCs), resulting in a second population of TILs, the first population of TILs obtainable by processing a tumor sample of a tumor resected from a cancer of a subject into a plurality of tumor fragments; wherein initiating the first amplification is performed in a vessel having a first gas permeable surface region, the initiating the first amplification being performed for a first period of time of about 1 to 7 days or about 1 to 8 days, obtaining a second population of TILs, the second population of TILs being greater in number than the first population of TILs;

(b) Performing a rapid second amplification by contacting the second TIL population with a cell culture medium of the second TIL population having additional IL-2, OKT-3, and APCs, producing a third TIL population, wherein the number of APCs in the rapid second amplification is at least twice the number of APCs in step (a), the rapid second amplification being performed for a second period of time of about 1 to 11 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, the rapid second amplification being performed in a container having a second gas permeable surface area;

(c) Collecting the therapeutic TIL population obtained from step (b); and

(D) Modifying a portion of the TILs at any time before or after collection in step (c) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

(a) Initiating a first expansion by culturing a first population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, and optionally Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed for a first period of about 1 to 7 days or about 1 to 8 days, obtaining a second population of TILs, the number of second population of TILs being greater than the first population of TILs;

(b) Performing a rapid second amplification by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3 and APC, producing a third population of TILs, wherein the rapid second amplification is performed for a second period of time of about 1 to 11 days, obtaining the third population of TILs, the third population of TILs being a therapeutic population of TILs;

(c) Collecting the therapeutic TIL population obtained from step (b); and

In some embodiments of this method, the cell culture medium in step (a) further comprises Antigen Presenting Cells (APCs), wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

In some embodiments of the methods provided herein, initiating the first amplification is divided into a first step and a second step, wherein the method further comprises performing the first step of initiating the first amplification by culturing the first population of TILs in a cell culture medium containing IL-2, producing TILs that are shed from the tumor fragments or samples, separating TILs remaining in the tumor fragments or samples from TILs shed from the tumor fragments or samples, optionally, digesting the tumor fragments or samples to produce tumor digests, performing the second step of initiating the first amplification by culturing the remaining TILs in the tumor fragments or samples or tumor digests in the cell culture medium, producing the second population of TILs.

(a) Obtaining and/or receiving a first population of TILs from a tumor sample obtained from one or more small biopsy, coarse needle biopsy, or needle aspiration biopsy of a tumor from a cancer of a subject by culturing the tumor sample in a first cell culture medium comprising IL-2 for about 3 days;

(b) Initiating a first expansion by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed in a vessel having a first gas permeable surface region, the initiating the first expansion being performed for a first period of about 7 or 8 days, obtaining the second population of TILs, the second population of TILs being greater in number than the first population of TILs;

(c) Generating a third population of TILs by performing a rapid second amplification by supplementing a second cell culture medium of the second population of TILs with additional IL-2, OKT-3 and APCs, wherein the number of APCs added in the rapid second amplification is at least twice the number of APCs added in step b), the rapid second amplification being performed for a second period of about 11 days, obtaining a third population of TILs, the third population of TILs being a therapeutic population of TILs, the rapid second amplification being performed in a container having a second gas permeable surface area;

(d) Collecting the therapeutic TIL population obtained from step (c);

(e) Transferring the collected TIL population from step (d) to an infusion bag; and

(F) Modifying a portion of the TILs at any time prior to transfer to the infusion bag in step (e) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

In another aspect, provided herein is a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, comprising:

(b) Initiating a first expansion by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed for a first period of about 7 or 8 days, obtaining a second population of TILs, the number of second population of TILs being greater than the first population of TILs;

(c) Performing a rapid second amplification by contacting the second population of TILs with a third cell culture medium comprising IL-2, OKT-3, and APC, producing a third population of TILs, wherein the rapid second amplification is performed for a second period of about 11 days, obtaining the third population of TILs, the third population of TILs being a therapeutic population of TILs;

(d) Collecting the therapeutic TIL population obtained from step (c); and

(E) Modifying a portion of the TILs at any time before or after collection in step (f) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

In some embodiments of the methods provided herein, the cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

In one aspect, provided herein is a method of expanding T cells comprising:

(a) Performing an initial first expansion of a first T cell population obtained from the donor by culturing the first T cell population to effect growth and initiate activation of the first T cell population;

(b) After the activation of the first T cell population initiated in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population to effect growth and enhance activation of the first T cell population, obtaining a second T cell population;

(c) Collecting a second T cell population; and

(D) Modifying a portion of the T cells at any time before or after collection in step (c) such that each of the modified T cells comprises an immunomodulatory composition associated with its surface membrane.

In another aspect, provided herein is a method of expanding T cells comprising:

(a) Performing an initial first expansion of a first T cell population from a tumor sample obtained from more than one small biopsy, coarse needle biopsy, or needle biopsy of a tumor in a donor by culturing the first T cell population to effect growth and initiate activation of the first T cell population;

(c) Collecting a second T cell population; and

(D) Modifying a portion of the T cells at any time before or after collection in step (e) such that each of the modified T cells comprises an immunomodulatory composition associated with its surface membrane.

In one aspect, provided herein is a method of amplifying Peripheral Blood Lymphocytes (PBLs) from peripheral blood, the method comprising the steps of:

(a) Obtaining a sample of Peripheral Blood Mononuclear Cells (PBMCs) from peripheral blood of a patient;

(b) Culturing the PBMCs in a culture comprising a first cell culture medium for a period of time selected from the group consisting of: about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, and about 14 days, thereby effecting expansion of Peripheral Blood Lymphocytes (PBLs) from said PBMCs, the first cell culture medium having a combination of IL-2, an anti-CD 3/anti-CD 28 antibody, and a first antibiotic;

(c) Collecting PBL from the culture in step (b); and

(D) Modifying a portion of the PBLs at any time before or after collection in step (c) such that each of the modified PBLs comprises an immunomodulatory composition associated with its surface membrane.

In some embodiments, the patient has been pre-treated with ibrutinib (ibrutinib) or another interleukin-2 inducible T cell kinase (ITK) inhibitor. In certain embodiments, the patient is refractory to treatment with ibrutinib or another ITK inhibitor.

In some embodiments, the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor moiety.

In exemplary embodiments, the one or more immunomodulators comprise one or more cytokines. In some embodiments, the one or more cytokines include IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNγ, TNFa, IFNα, IFNβ, GM-CSF or GCSF or variants thereof.

In some embodiments, the one or more cytokines include IL-2. In some embodiments, IL-2 is human IL-2. In an exemplary embodiment, human IL-2 has the sequence set forth in SEQ ID NO: 272.

In some embodiments, more than one cytokine including IL-12. In certain embodiments, IL-12 comprises a human IL-12p35 subunit linked to a human IL-12p40 subunit. In certain embodiments, the human IL-12p35 subunit has the sequence of SEQ ID NO:267, the human IL-12p40 subunit has the amino acid sequence of SEQ ID NO: 268.

In some embodiments, the one or more cytokines include IL-15. In some embodiments, IL-15 is human IL-15. In an exemplary embodiment, human IL-15 has the sequence set forth in SEQ ID NO: 258.

In some embodiments, the one or more cytokines include IL-18. In some embodiments, IL-18 is human IL-18. In certain embodiments, human IL-18 has the sequence set forth in SEQ ID NO:269 or SEQ ID NO:270, and a sequence of amino acids.

In some embodiments, the one or more cytokines include IL-21. In certain embodiments, IL-21 is human IL-21. In some embodiments, human IL-21 has the sequence of SEQ ID NO: 251.

In some embodiments, the one or more cytokines include IL-15 and IL-21. In some embodiments, IL-15 is human IL-15, IL-21 is human IL-21. In certain embodiments, human IL-15 has the amino acid sequence of SEQ ID NO:258, human IL-21 has the amino acid sequence of SEQ ID NO:271, and a fragment thereof.

In some embodiments, the one or more immunomodulators comprise a CD40 agonist. In certain embodiments, the CD40 agonist is an anti-CD 40 binding domain or CD40L. In an exemplary embodiment, the CD40 agonist is a CD40 binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, VH and VL of the CD40 binding domain are selected from the following: a) Has the sequence of SEQ ID NO:274 and VH having the amino acid sequence of SEQ ID NO:275, VL of an amino acid sequence; b) Has the sequence of SEQ ID NO:277 and VH having the amino acid sequence of SEQ ID NO:278, a VL of the amino acid sequence of 278; c) Has the sequence of SEQ ID NO:280 and a VH having the amino acid sequence of SEQ ID NO:281 amino acid sequence VL; and d) having the sequence of SEQ ID NO:283 and VH having the amino acid sequence of SEQ ID NO:284, VL of the amino acid sequence of seq id no. In an exemplary embodiment, the CD40 binding domain is an scFv.

In some embodiments, the CD40 agonist is a polypeptide having the amino acid sequence of SEQ ID NO:273, human CD40L of the amino acid sequence.

In some embodiments, one or more membrane-anchored immunomodulatory fusion proteins are independently according to the following formula from N-terminus to C-terminus: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety.

In some embodiments, the cell membrane anchor portion comprises a CD8a transmembrane-intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In an exemplary embodiment, the cell membrane anchor portion comprises a cytokine B7-1 transmembrane domain. In some embodiments, the cell membrane anchor portion has the amino acid sequence of SEQ ID NO: 239.

In some embodiments, the immunomodulatory composition comprises two or more different membrane-anchored immunomodulatory fusion proteins, wherein each of the different membrane-anchored immunomodulatory fusion proteins each comprises a different immunomodulatory agent. In some embodiments, the different immunomodulatory agent is selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF, GCSF, or variants thereof, and CD40 agonists. In some embodiments, the different immunomodulatory agent is selected from: IL-12 and IL-15, IL-15 and IL-18, IL-15 and IL-21, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12.

In some embodiments, the modified TIL comprises a first membrane-anchored immunomodulatory fusion protein and a second membrane-anchored immunomodulatory fusion protein.

In some embodiments, the first membrane-anchored immunomodulatory fusion protein comprises IL-15 and the second membrane-anchored immunomodulatory fusion protein comprises IL-21.

In exemplary embodiments, the expression of the first membrane-anchored immunomodulatory fusion protein and the second membrane-anchored immunomodulatory fusion protein is controlled by the NFAT promoter in the modified TIL.

In exemplary embodiments, one or more membrane-anchored immunomodulatory fusion proteins are independently according to the following formula from N-terminus to C-terminus: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety. In some embodiments, IA is a cytokine. In an exemplary embodiment, the IA is selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof. In some embodiments, the IA is IL-2. In certain embodiments, the IA is IL-12. In some embodiments, the IA is IL-15. In certain embodiments, the IA is IL-21.

For example, one or more membrane-anchored immunomodulatory fusion proteins are independently according to the following formula from N-terminus to C-terminus: S1-IA1-L1-C1-L2-S2-IA2-L3-C2, wherein S1 and S2 are each independently a signal peptide, IA1 and IA2 are each independently an immunomodulator, L1-L3 are each independently a linker, and C1 and C2 are each independently a cell membrane anchor moiety. In some embodiments, S1 is the same as S2. In certain embodiments, C1 is the same as C2. In some embodiments, L2 is a cleavable linker. In exemplary embodiments, L2 is a furin (furin) cleavable linker. In some embodiments, IA1 and IA2 are each independently cytokines.

In some embodiments, IA1 and IA2 are each independently selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof. In some embodiments, IA1 and IA2 are each independently selected from IL-2 and IL-12, with the proviso that one of IA1 and IA2 is IL-2 and the other is IL-12. In some embodiments, IA1 and IA2 are each independently selected from: IL-15 and IL-21, with the proviso that one of IA1 and IA2 is IL-15 and the other is IL-21.

In certain embodiments, the modification comprises introducing a heterologous nucleic acid encoding a fusion protein into a portion of the TIL and expressing the fusion protein on the surface of the modified TIL.

In certain embodiments, the modification comprises introducing a heterologous nucleic acid encoding a fusion protein into a portion of the TIL and expressing the fusion protein on the surface of the modified TIL. In some embodiments, the heterologous nucleic acid is introduced into the genome of the modified TIL using one or more methods selected from the group consisting of: CRISPR method, TALE method, zinc finger method and combinations thereof.

In some embodiments, the immunomodulatory composition comprises a fusion protein comprising one or more immunomodulatory agents linked to a TIL surface antigen binding domain. In some embodiments, the one or more immunomodulators comprise one or more cytokines. In some embodiments, the one or more cytokines include IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNγ, TNFa, IFNα, IFNβ, GM-CSF or GCSF or variants thereof. In some embodiments, more than one cytokine including IL-12. In certain embodiments, the one or more cytokines include IL-15. In some embodiments, the one or more cytokines include IL-21. In certain embodiments, the TIL surface antigen binding domain comprises an antibody heavy chain variable domain and a light chain variable domain. In some embodiments, the TIL surface antigen binding domain comprises an antibody or fragment thereof. In some embodiments, the TIL surface antigen binding domain has an affinity ：CD45、CD4、CD8、CD3、CDlla、CDllb、CDllc、CD18、CD25、CD127、CD19、CD20、CD22、HLA-DR、CD197、CD38、CD27、CD196、CXCR3、CXCR4、CXCR5、CD84、CD229、CCR1、CCR5、CCR4、CCR6、CCR8、CCR10、CD 16、CD56、CD 137、OX40 or GITR for more than one of the following TIL surface antigens. In certain embodiments, the modification comprises incubating the fusion protein with a portion of the TIL under conditions that allow the fusion protein to bind to the portion of the TIL.

In some embodiments, the immunomodulatory composition comprises a nanoparticle comprising a plurality of immunomodulatory agents. In some embodiments, the plurality of immunomodulatory agents are covalently linked together by a degradable linker. In certain embodiments, the nanoparticle comprises at least one polymer, cationic polymer, or cationic block copolymer on the surface of the nanoparticle. In some embodiments, the one or more cytokines include IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNγ, TNFa, IFNα, IFNβ, GM-CSF or GCSF or variants thereof. In certain embodiments, the one or more cytokines include IL-12. In some embodiments, the one or more cytokines include IL-15. In some embodiments, the one or more cytokines include IL-21. In some embodiments, the nanoparticle is a liposome, a protein nanogel, a nucleotide nanogel, a polymer nanoparticle, or a solid nanoparticle. In some embodiments, the nanoparticle is a nanogel. In certain embodiments, the nanoparticle further comprises an antigen binding domain that binds more than one of the following antigens: CD45, CDlla (integrin α -L), CD18 (integrin β -2), CD1lb, CD1lc, CD25, CD8 or CD4. In some embodiments, modifying comprises attaching the immunomodulatory composition to a surface of a portion of the TIL.

In certain embodiments of the methods provided herein, the TIL from the first amplification or the TIL from the second amplification, or both, are modified. In certain embodiments, the TIL from the initial first amplification or the TIL from the rapid second amplification, or both, are modified.

In some embodiments of the methods provided herein, the modification is performed after the first amplification and before the second amplification. In some embodiments, the modification is performed after initiation of the first amplification and before rapid second amplification, or at both time points. In certain embodiments, the modification is performed after the second amplification. In some embodiments, the modification is performed after the rapid second amplification. In some embodiments, the modification is performed after collection.

In certain embodiments, the first amplification is performed for a period of about 11 days. In some embodiments, the initiating the first amplification is performed for a period of about 11 days.

In some embodiments of the methods provided herein, in the first amplification, the IL-2 is present in the cell culture medium at an initial concentration of between 1000IU/mL and 6000 IU/mL. In certain embodiments, IL-2 is present in the cell culture medium in which the first expansion is initiated at an initial concentration of between 1000IU/mL and 6000 IU/mL.

In some embodiments, in the second amplification step, IL-2 is present at an initial concentration of between 1000IU/mL and 6000IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL. In some embodiments, in the rapid second amplification step, IL-2 is present at an initial concentration of between 1000IU/mL and 6000IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, the first amplification is performed using a gas-permeable container. In certain embodiments, the initial first amplification is performed using a gas-permeable container. In some embodiments, the second amplification is performed using a gas-permeable container. In certain embodiments, the rapid second amplification is performed using a gas-permeable container.

In some embodiments of the methods provided herein, the first expanded cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof. In certain embodiments, the cell culture medium that initiates the first expansion further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof. In some embodiments, the second expanded cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof. In certain embodiments, the rapid second expansion cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof.

In some embodiments of the methods of treatment provided herein, the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering TIL to the patient. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day for two days, followed by fludarabine (fludarabine) at a dose of 25mg/m ²/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day and fludarabine was administered at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for three days. In certain embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day and fludarabine was administered at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for one day. In certain embodiments, cyclophosphamide is administered with mesna (mesna).

In some embodiments of the methods of treatment provided herein, the method further comprises the step of beginning the treatment of the patient with the IL-2 regimen the next day after the administration of TIL to the patient. In some embodiments of the methods of treatment provided herein, the method further comprises the step of beginning treatment of the patient with the IL-2 regimen on the same day as the administration of the TIL to the patient. In some embodiments, the IL-2 regimen is a high dose IL-2 regimen comprising 600,000 or 720,000IU/kg of aldesleukin (aldesleukin) or a biological analog or variant thereof, which is administered in the form of an intravenous infusion of 15 minute bolus injections every eight hours until tolerated.

In some embodiments of the methods provided herein, a therapeutically effective population of TILs comprising about 2.3 x 10 ¹⁰ to about 13.7 x 10 ¹⁰ TILs is administered.

In some embodiments of the methods provided herein, initiating the first amplification and rapidly second amplification are performed for a period of time less than 21 days. In some embodiments, initiating the first amplification and rapidly second amplification are performed for a period of time less than 16 or 17 days. In certain embodiments, the initial first amplification is performed for a period of time less than 7 or 8 days. In some embodiments, the rapid second amplification is performed for a period of time less than 11 days.

In some embodiments of the methods provided herein, the first amplification in step (c) and the second amplification in step (d) are each separately performed over a period of 11 days. In some embodiments of the methods provided herein, steps (a) through (f) are performed for about 10 days to about 22 days.

In some embodiments of the methods provided herein, the modified TIL further comprises a genetic modification that results in silencing or reduction of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. In some embodiments, the one or more immune checkpoint genes are selected from ：PD-1、CTLA-4、LAG-3、HAVCR2(TIM-3)、Cish、TGFβ、PKA、CBL-B、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、BTLA、CD160、TIGIT、TET2、CD96、CRTAM、LAIR1、SIGLEC7、SIGLEC9、CD244、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT1、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2、GUCY1B3、TOX、SOCS1、ANKRD11 and BCOR. In certain embodiments, the one or more immune checkpoint genes are selected from: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), cish, TGF beta and PKA.

In some embodiments, the modified TIL further comprises a genetic modification that results in an increase in expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population, the one or more immune checkpoint genes selected from the group consisting of: CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1. In certain embodiments, the genetic modification is performed using a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at more than one immune checkpoint gene. In some embodiments, the genetic modification is performed using one or more methods selected from the group consisting of: CRISPR method, TALE method, zinc finger method and combinations thereof. In certain embodiments, the gene modification is performed using a CRISPR method. In some embodiments, the CRISPR method is a CRISPR/Cas9 method. In certain embodiments, the TALE method is used for genetic modification. In some embodiments, the genetic modification is performed using a zinc finger approach.

In some embodiments, the modified TIL is modified to transiently express the immunomodulatory composition on the cell surface. In some embodiments, the immunomodulatory composition comprises one or more membrane anchored immunomodulatory fusion proteins, wherein each fusion protein comprises one or more immunomodulatory agents and a membrane anchor moiety.

In some embodiments, the one or more cytokines include IL-21. In certain embodiments, IL-21 is human IL-21. In some embodiments, human IL-21 has the sequence of SEQ ID NO:271, and a fragment thereof.

In some embodiments, the one or more immunomodulators comprise a CD40 agonist. In certain embodiments, the CD40 agonist is an anti-CD 40 binding domain or CD40L. In an exemplary embodiment, the CD40 agonist is a CD40 binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, VH and VL of the CD40 binding domain are selected from: a) Has the sequence of SEQ ID NO:274 and VH having the amino acid sequence of SEQ ID NO:275, VL of an amino acid sequence; b) Has the sequence of SEQ ID NO:277 and VH having the amino acid sequence of SEQ ID NO:278, a VL of the amino acid sequence of 278; c) Has the sequence of SEQ ID NO:280 and a VH having the amino acid sequence of SEQ ID NO:281 amino acid sequence VL; and d) having the sequence of SEQ ID NO:283 and VH having the amino acid sequence of SEQ ID NO:284, VL of the amino acid sequence of seq id no. In an exemplary embodiment, the CD40 binding domain is an scFv.

In some embodiments, the CD40 agonist is a polypeptide having the amino acid sequence of SEQ ID NO:273, human CD40L of the amino acid sequence. In some embodiments, the membrane-anchored immunomodulatory fusion protein is according to the formula: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety.

In some embodiments, the cell membrane anchor portion comprises a CD8a transmembrane-intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In an exemplary embodiment, the cell membrane anchor portion comprises a B7-1 transmembrane domain. In some embodiments, the cell membrane anchor portion has the amino acid sequence of SEQ ID NO: 239.

In some embodiments, the immunomodulatory composition comprises two or more different membrane-anchored immunomodulatory fusion proteins, wherein each of the different membrane-anchored immunomodulatory fusion proteins each comprises a different immunomodulatory agent. In some embodiments, the different immunomodulatory agent is selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF, GCSF, or variants thereof, and CD40 agonists. In some embodiments, the different immunomodulatory agent is selected from: IL-12 and IL-15, IL-15 and IL-18, CD40L, IL-15 and IL-21, and IL-15, and IL-2 and IL-12.

In some embodiments, the modified TIL is modified by transfecting the TIL with a nucleic acid encoding a fusion protein comprising one or more immunomodulators and a cell membrane anchor moiety to transiently express the fusion protein on the cell surface. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the TIL is transfected with mRNA by electroporation. In some embodiments, the TIL is transfected with mRNA by electroporation after the first amplification and before the second amplification. In some embodiments, TIL is transfected with mRNA by electroporation prior to the first amplification. In some embodiments, the method further comprises: prior to transfection of TIL with mRNA, TIL was activated by incubation with an anti-CD 3 agonist. In some embodiments, the anti-CD 3 agonist is OKT-3. In some embodiments, the TIL is activated by incubating the TIL with an anti-CD 3 agonist for about 1 to 3 days prior to transfection of the TIL with mRNA.

In some embodiments, the modified TIL is transfected with a nucleic acid encoding a fusion protein by temporarily disrupting the cell membrane of the TIL using a microfluidic device, thereby effecting transfection of the nucleic acid.

In some embodiments, an artificial antigen presenting cell (aAPC) is used in place of an APC. In some embodiments, the aAPCs include cells that express HLA-A/B/C, CD, CD80, ICOS-L, and CD58. In some embodiments, the aapcs comprise MOLM-14 cells. In some embodiments, the aapcs comprise MOLM-13 cells. In some embodiments, the aAPC comprises a MOLM-14 cell that endogenously expresses HLA-A/B/C, CD64, CD80, ICOS-L, and CD58. In some embodiments, the aAPC comprises a MOLM-14 cell that endogenously expresses HLA-A/B/C, CD64, CD80, ICOS-L, and CD58, and the MOLM-14 cell is permanently genetically edited to express CD86. In some embodiments, MOLM-14 cells are transduced with more than one viral vector, wherein the more than one viral vector comprises a nucleic acid sequence encoding CD86 and a nucleic acid sequence encoding 4-1BBL, and the MOLM-14 cells express CD86 and 4-1BBL. In some embodiments, aapcs are transiently genetically edited to transiently express an immunomodulatory composition comprising an immunomodulatory fusion protein on a cell surface. In some embodiments, aapcs transiently express an immunomodulatory fusion protein on the cell surface comprising a membrane anchor fused to a cytokine. In some embodiments, aapcs transiently express a membrane anchor on the cell surface fused to a cytokine selected from the group consisting of: IL-2, IL-7, IL-10, IL-12, IL-15 and IL-21. In some embodiments, aapcs transiently express a membrane anchor on the cell surface fused to a cytokine selected from the group consisting of: IL-2, IL-12, IL-15 and IL-21. In some embodiments, aapcs transiently express a membrane anchor on the cell surface fused to a cytokine selected from the group consisting of: IL-12, IL-15 and IL-21.

In some embodiments, the modified TIL is genetically modified to express an immunomodulatory composition on the surface of a cell. In some embodiments, the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor moiety. In some embodiments, one or more membrane-anchored immunomodulatory fusion proteins comprise IL-2. In certain embodiments, one or more membrane-anchored immunomodulatory fusion proteins comprise IL-15. In exemplary embodiments, one or more membrane-anchored immunomodulatory fusion proteins comprise IL-18. In some embodiments, more than one membrane anchored immunomodulatory fusion protein comprises IL-21.

In certain embodiments, the modified TIL comprises a first membrane-anchored immunomodulatory fusion protein and a second membrane-anchored immunomodulatory fusion protein. In some embodiments, the first membrane-anchored immunomodulatory fusion protein comprises IL-15 and the second membrane-anchored immunomodulatory fusion protein comprises IL-21. In some embodiments, the expression of the first membrane anchored immunomodulatory fusion protein and the second immunomodulatory fusion protein is controlled by the NFAT promoter in the modified TIL.

In some embodiments, one or more membrane-anchored immunomodulatory fusion proteins are independently according to the following formula from N-terminus to C-terminus: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety. In some embodiments, IA is a cytokine. In an exemplary embodiment, the IA is selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof. In some embodiments, the IA is IL-2. In certain embodiments, the IA is IL-12. In some embodiments, the IA is IL-15. In certain embodiments, the IA is IL-21. In some embodiments, L is a CD8a transmembrane-intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In certain embodiments, L is a B7-1 transmembrane domain. In some embodiments, L has the amino acid sequence of SEQ ID NO: 239.

In exemplary embodiments, one or more membrane-anchored immunomodulatory fusion proteins are independently according to the following formula from N-terminus to C-terminus: S1-IA1-L1-C1-L2-S2-IA2-L3-C2, wherein S1 and S2 are each independently a signal peptide, IA1 and IA2 are each independently an immunomodulator, L1-L3 are each independently a linker, and C1 and C2 are each independently a cell membrane anchor moiety. In some embodiments, S1 is the same as S2. In an exemplary embodiment, C1 is the same as C2. In some embodiments, L2 is a cleavable linker. In certain embodiments, L2 is a furin cleavable linker.

In some embodiments, IA1 and IA2 are each independently cytokines. In some embodiments, IA1 and IA2 are each independently selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof. In some embodiments, IA1 and IA2 are each independently selected from IL-2 and IL-12, with the proviso that one of IA1 and IA2 is IL-2 and the other is IL-12. In some embodiments, IA1 and IA2 are each independently selected from: IL-15 and IL-21, with the proviso that one of IA1 and IA2 is IL-15 and the other is IL-21.

In exemplary embodiments, C1 and C2 are each independently a CD8a transmembrane-intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In some embodiments, C1 and C2 are each a B7-1 transmembrane domain. In some embodiments, C1 and C2 each have the amino acid sequence of SEQ ID NO: 239.

In certain embodiments, the modified TIL expresses one or more membrane-anchored immunomodulatory fusion proteins under the control of the NFAT promoter. In some embodiments, the modified TIL is transduced with a retroviral vector to express one or more membrane anchored immunomodulatory fusion proteins. In some embodiments, the modified TIL is transduced with a lentiviral vector to express one or more membrane anchored immunomodulatory fusion proteins.

Drawings

Fig. 1: an exemplary Gen 2 (process 2A) chart provides an overview of steps a through F.

Fig. 2A to 2C: process flow diagram of an embodiment of Gen 2 (process 2A) for TIL manufacture.

Fig. 3: a graphical representation of an example of a cryopreserved TIL exemplary manufacturing process (about 22 days) is shown.

Fig. 4: a graph showing an example of Gen 2 (process 2A, i.e., a 22 day process for TIL manufacture) is shown.

Fig. 5: comparison table of procedure 1C and steps a to F of an exemplary embodiment of Gen 2 (procedure 2A) for TIL manufacture.

Fig. 6: detailed comparison of the example of procedure 1C and the example of Gen 2 (procedure 2A) for TIL manufacture.

Fig. 7: exemplary Gen 3 type TIL manufacturing process.

Fig. 8A to 8D: a) A comparison between examples of the 2A process (about 22 day process) and the Gen 3 process (about 14 to 16 day process) for TIL manufacture is shown. B) An exemplary Gen 3 process chart provides an overview of steps a through F (about 14 to 16 days of process). C) A chart of three exemplary Gen 3 processes is provided summarizing steps a through F (about 14 day or 16 day process) for each of the three process variants. D) An exemplary modified Gen-like 2 procedure provides an overview of steps a through F (about 22 day procedure).

Fig. 9: an experimental flow chart providing comparability between Gen 2 (process 2A) and Gen 3 processes.

Fig. 10: a comparison between various Gen 2 (process 2A) and Gen 3.1 process examples is shown.

Fig. 11: tables describing various features of embodiments of the Gen 2, gen 2.1 and Gen 3.0 processes.

Fig. 12: summary of the medium conditions of the examples of the Gen 3 process (referred to as Gen 3.1).

Fig. 13: tables describing various features of embodiments of the Gen 2, gen 2.1 and Gen 3.0 processes.

Fig. 14: tables comparing various features of examples of Gen 2 and Gen 3.0 processes.

Fig. 15: a table of media uses in various embodiments of the described amplification process is provided.

Fig. 16: schematic of an exemplary embodiment of the Gen 3 process (16 day process).

Fig. 17: schematic of an exemplary embodiment of a method for amplifying T cells from hematopoietic malignancies using a Gen 3 amplification platform.

Fig. 18: structures I-A and I-B are provided. Cylinders refer to individual polypeptide binding domains. Structures I-a and I-B comprise three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein by an IgG1-Fc (comprising CH3 and CH2 domains) which subsequently links the two trivalent proteins together by disulfide bonds (small oblong), thereby stabilizing the structure and providing an agonist capable of bringing together the intracellular signaling domains of the six receptors and the signaling proteins to form a signaling complex. The TNFRSF binding domain represented as a cylinder may be an scFv domain comprising, for example, V _H and V _L chains connected by a linker, which may comprise hydrophilic residues and Gly and Ser sequences providing flexibility and Glu and Lys providing solubility.

Fig. 19: schematic of an exemplary embodiment of the Gen 3 process (16 day process).

Fig. 20: a process overview of an exemplary embodiment of the Gen 3.1 process (16 day process) is provided.

Fig. 21: schematic of an exemplary embodiment of a Gen 3.1 test procedure (16 to 17 day procedure).

Fig. 22: schematic of an exemplary embodiment of the Gen 3 process (16 day process).

Fig. 23: comparison table of exemplary Gen 2 and exemplary Gen 3 processes.

Fig. 24: gen 3 procedure (16-17 day procedure) a schematic of an exemplary embodiment of a timeline was prepared.

Fig. 25: schematic of an exemplary embodiment of the Gen 3 process (14 to 16 day process).

Fig. 26A to 26B: schematic of an exemplary embodiment of the Gen 3 process (16 day process).

Fig. 27: schematic of an exemplary embodiment of the Gen 3 process (16 day process).

Fig. 28: comparison of examples of Gen 2, gen 2.1 and Gen 3 procedure (16 day procedure).

Fig. 29: comparison of examples of Gen 2, gen 2.1 and Gen 3 procedure (16 day procedure).

Fig. 30: gen 3 example Components.

Fig. 31: gen 3 example flow chart comparison (Gen 3.0, gen 3.1 control, gen 3.1 test).

Fig. 32: the components of an exemplary embodiment of the Gen 3 process (16 to 17 day process) are shown.

Fig. 33: and checking an acceptance criterion table.

Fig. 34: description of some embodiments of TIL manufacturing processes including electroporation steps used with gene editing processes (including TALEN, zinc finger nucleases, and CRISPR methods as described herein).

Fig. 35: description of an embodiment of a TIL manufacturing process comprising an electroporation step used with a gene editing process (including TALEN, zinc finger nucleases, and CRISPR methods as described herein).

Fig. 36: exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein.

Fig. 37: exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein.

Fig. 38: summary of studies for assessing expression and signaling of pre-REP TILs for membrane-bound IL-15/IL-21 transduction.

Fig. 39: summary of studies for assessing expression of mIL-15/IL21 and CD8 and CD 4T cell subsets in mIL-15/IL-21 transduced REP TIL.

Fig. 40: summary of studies used to assess the phenotype of mIL-15/IL-21 transduced CD8+ REP TILs.

Fig. 41: summary of studies used to assess the phenotype of mIL-15/IL-21 transduced CD4+.

Brief description of the sequence Listing

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

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

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

SEQ ID NO:4 is the amino acid sequence of aldesleukin (aldesleukin).

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

SEQ ID NO:6 is the amino acid sequence of nevalknob Jin (nemvaleukin alfa).

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

SEQ ID NO:8 is a mucin domain polypeptide.

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

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

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

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

SEQ ID NO:13 is IL-2 sequence.

SEQ ID NO:14 is IL-2 mutein sequence.

SEQ ID NO:15 is IL-2 mutein sequence.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:36 is the V _L chain.

SEQ ID NO:37 is the light chain.

SEQ ID NO:38 is the light chain.

SEQ ID NO:39 is a light chain.

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

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

SEQ ID NO:42 is the heavy chain of the 4-1BB agonist monoclonal antibody Wu Tumu mab (utomilumab) (PF-05082566).

SEQ ID NO:43 is the light chain of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:44 is the heavy chain variable region (V _H) of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:45 is the light chain variable region (V _L) of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Wu Tumu (PF-05082566).

SEQ ID NO:49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody Wu Tumu mab (PF-05082566).

SEQ ID NO:52 is the heavy chain of the 4-1BB agonist monoclonal antibody Wu Ruilu mab (urelumab) (BMS-663513).

SEQ ID NO:53 is the light chain of the 4-1BB agonist monoclonal antibody Wu Ruilu mab (BMS-663513).

SEQ ID NO:54 is the heavy chain variable region (V _H) of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

SEQ ID NO:55 is the light chain variable region (V _L) of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

SEQ ID NO:56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

SEQ ID NO:57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

SEQ ID NO:58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

SEQ ID NO:59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

SEQ ID NO:60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

SEQ ID NO:61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody Wu Ruilu (BMS-663513).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:84 is the light chain variable region (V _L) of the 4-1BB agonist antibody H39E 3-2.

SEQ ID NO:85 is the amino acid sequence of human OX 40.

SEQ ID NO:86 is the amino acid sequence of murine OX 40.

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:97 is the heavy chain of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:98 is the light chain of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:99 is the heavy chain variable region (V _H) of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:100 is the light chain variable region (V _L) of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:101 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:102 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:103 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:104 is the light chain CDR1 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:105 is the light chain CDR2 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:106 is the light chain CDR3 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:107 is the heavy chain of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:108 is the light chain of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:109 is the heavy chain variable region (V _H) of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:110 is the light chain variable region (V _L) of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:111 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:112 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:113 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:114 is the light chain CDR1 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:115 is the light chain CDR2 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:116 is the light chain CDR3 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:117 is the heavy chain variable region (V _H) of the OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO:118 is the light chain variable region (V _L) of the OX40 agonist monoclonal antibody Hu 119-122.

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

SEQ ID NO:120 is the heavy chain CDR2 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO:121 is the heavy chain CDR3 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO:122 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO:123 is the light chain CDR2 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO:124 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO:125 is the heavy chain variable region (V _H) of the OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:126 is the light chain variable region (V _L) of the OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:127 is the heavy chain CDR1 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:128 is the heavy chain CDR2 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:129 is the heavy chain CDR3 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:130 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:131 is the light chain CDR2 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:132 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:133 is the amino acid sequence of OX40 ligand (OX 40L).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:153 are the heavy chain variable regions of humanized OX40 agonist monoclonal antibodies (V _H).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:170 is the heavy chain variable region (V _H) amino acid sequence of the PD-1 inhibitor pamil mab.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:187 are the light chain CDR3 amino acid sequences of the PD-L1 inhibitor dewaruzumab.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:230 is the heavy chain variable region (V _H) amino acid sequence of the CTLA-4 inhibitor Zeff mab.

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

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

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

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

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

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

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

SEQ ID NO:238 is the CD8a transmembrane domain.

SEQ ID NO:239 is B7-1 transmembrane-intracellular domain.

SEQ ID NO: 240-245 are exemplary glycine-serine linkers suitable for use in immunomodulatory fusion proteins described herein.

SEQ ID NO:246 are exemplary linkers suitable for use with the immunomodulatory fusion proteins described herein.

SEQ ID NO:247 is the C-terminal sequence of the 2A peptide.

SEQ ID NO:248 is porcine teschovirus type 1 type 2A peptide.

SEQ ID NO:249 is the horseshoe-shaped rhinitis virus 2A peptide.

SEQ ID NO:250 is hand-foot-mouth disease virus 2A peptide.

SEQ ID NO:251 is an exemplary furin cleavable 2A peptide.

SEQ ID NO:252 and 253 are human IgE signal peptide sequences. SEQ ID NO:254 is the human IL-2 signal peptide sequence.

SEQ ID NO:255 is the 6 XNFAT IL-2 minimal promoter.

SEQ ID NO:256 is an NFAT response element.

SEQ ID NO:557 is the human IL-2 promoter sequence.

SEQ ID NO:258 is human IL-15 (N72D mutant).

SEQ ID NO:259 is the human IL-15R-alpha-Su/Fc domain.

SEQ ID NO:260 is human IL-15R-alpha-Su (65 aa truncated extracellular domain).

SEQ ID NO:261 is human IL-15 isomer (isosporm) 2.

SEQ ID NO:262 is human IL-15 isomer 1.

SEQ ID NO:263 is human IL-15 (without signal peptide).

SEQ ID NO:264 is human IL-15R- α (85 aa truncated extracellular domain).

SEQ ID NO:265 is human IL-15R-alpha (182 aa truncated extracellular domain).

SEQ ID NO:266 is human IL-15R-alpha.

SEQ ID NO:267 is human IL-12p35 subunit.

SEQ ID NO:268 is the human IL-12p40 subunit.

SEQ ID NO:269 is human IL-18.

SEQ ID NO:270 is a human IL-18 variant.

SEQ ID NO:271 is human IL-21.

SEQ ID NO:272 is human IL-2.

SEQ ID NO:273 is human CD40L.

SEQ ID NO:274 is agonistic anti-human CD40 VH (sotigalizumab (Sotigalimab)).

SEQ ID NO:275 is an agonistic anti-human CD40 VL (sotigalizumab).

SEQ ID NO:276 is an agonistic anti-human CD40 scFv (sotigalizumab).

SEQ ID NO:277 is an agonistic anti-human CD40 VH (daclizumab (Dacetuzumab)).

SEQ ID NO:278 is an agonistic anti-human CD40 VL (daclizumab).

SEQ ID NO:279 is an agonistic anti-human CD40 scFv (daclizumab).

SEQ ID NO:280 is agonistic anti-human CD40 VH (Lu Katuo bead (Lucatutuzumab)).

SEQ ID NO:281 is an agonistic anti-human CD40 VL (Lu Katuo bead mab).

SEQ ID NO:282 is an agonistic anti-human CD40 scFv (Lu Katuo bead mab).

SEQ ID NO:283 is an agonistic anti-human CD40 VH (brumab (Selicrelumab)).

SEQ ID NO:284 is an agonistic anti-human CD40 VL (seluzumab).

SEQ ID NO:285 is an agonistic anti-human CD40 scFv (seluzumab).

SEQ ID NO:286 is the target PD-1 sequence.

SEQ ID NO:287 is the target PD-1 sequence.

SEQ ID NO:288 is the repeated PD-1 left repeat sequence.

SEQ ID NO:289 is a repetitive PD-1 right repetitive sequence.

SEQ ID NO:290 is a repeat PD-1 left repeat.

SEQ ID NO:291 is the repeat PD-1 right repeat.

SEQ ID NO:292 is PD-1 left TALEN nuclease sequence.

SEQ ID NO:293 is PD-1 right TALEN nuclease sequence.

SEQ ID NO:294 is the PD-1 left TALEN nuclease sequence.

SEQ ID NO:295 is PD-1 right TALEN nuclease sequence.

SEQ ID NO:296 is a nucleotide sequence encoding SEQ ID NO:328 to tether (tethered) IL-15.

SEQ ID NO:297 is the nucleotide sequence encoding SEQ ID NO: is tethered to the nucleic acid sequence of the IL-21 fusion protein.

SEQ ID NO:298 is a nucleotide sequence encoding SEQ ID NO:328 and SEQ ID NO:331 tethered IL-21 fusion protein.

SEQ ID NO:299 is a polypeptide encoding SEQ ID NO:303 to tether IL-12 fusion proteins. The nucleic acid sequence comprises the NFAT promoter.

SEQ ID NO:300 is the sequence encoding SEQ ID NO:328 to tether IL-15 fusion protein. The nucleic acid sequence comprises the NFAT promoter.

SEQ ID NO:301 is a nucleotide sequence encoding SEQ ID NO: XX tethered IL-21 fusion protein nucleic acid sequence. The nucleic acid sequence comprises the NFAT promoter.

SEQ ID NO:302 is a nucleic acid encoding SEQ ID NO:328 and SEQ ID NO:331 tethered IL-21 fusion protein. The nucleic acid sequence comprises the NFAT promoter.

SEQ ID NO:303 is an exemplary tethered IL-12 (tethered IL-12-Lr1-Ar 2) amino acid sequence.

SEQ ID NO:304 is a polypeptide encoding SEQ ID NO:303 to tether IL-12.

SEQ ID NO:305 is an exemplary tethered IL-18 (tethered IL-18-Lr1-Ar 2) amino acid sequence.

SEQ ID NO:306 is a nucleic acid encoding SEQ ID NO:305 to tether IL-18.

SEQ ID NO:307 is the amino acid sequence of an exemplary tethered IL-18 variant (tethered DR-IL-18 (6-27 variant) -Lr1-Ar 2).

SEQ ID NO:308 is the sequence encoding SEQ ID NO:307 to tether IL-18 variants.

SEQ ID NO:309 is an exemplary tethered IL-12/IL-15 amino acid sequence.

SEQ ID NO:310 is a polypeptide encoding SEQ ID NO:309 to tether IL-12/IL-15.

SEQ ID NO:311 is an exemplary tethered IL-18/IL-15 amino acid sequence.

SEQ ID NO:312 is a polypeptide encoding SEQ ID NO:311 to tether IL-18/IL-15 nucleic acid sequence.

SEQ ID NO:313 is an exemplary tethered anti-CD 40scFV (APX 005M) amino acid sequence.

SEQ ID NO:314 is the sequence encoding SEQ ID NO:313 to tether an anti-CD 40scFV (APX 005M).

SEQ ID NO:315 is an exemplary tethered anti-CD 40scFV (daclizumab) amino acid sequence.

SEQ ID NO:316 is the sequence encoding SEQ ID NO:315 to a nucleic acid sequence of a tethered anti-CD 40scFV (daclizumab).

SEQ ID NO:317 is the amino acid sequence of an exemplary tethered anti-CD 40scFV (Lu Katuo bead mab).

SEQ ID NO:318 is the sequence encoding SEQ ID NO:317 to the nucleic acid sequence of a tethered anti-CD 40scFV (Lu Katuo bead mab).

SEQ ID NO:319 is an exemplary tethered anti-CD 40scFV (seluzumab) amino acid sequence.

SEQ ID NO:320 is the sequence encoding SEQ ID NO:319 to a tethered anti-CD 40scFV (brucella).

SEQ ID NO:321 is the sequence encoding SEQ ID NO:273 CD40L nucleic acid sequence.

SEQ ID NO:322 is an exemplary tethered CD40L/IL-15 amino acid sequence.

SEQ ID NO:323 is the sequence encoding SEQ ID NO:311 to tether CD 40L/IL-15.

SEQ ID NO:324 is an amino acid sequence of an exemplary tethered IL-2.

SEQ ID NO:325 is a polypeptide encoding SEQ ID NO:313 to tether IL-2.

SEQ ID NO:326 is an exemplary tethered IL-12 amino acid sequence.

SEQ ID NO:327 is a polypeptide encoding SEQ ID NO:315 to tether IL-12.

SEQ ID NO:328 is an exemplary tethered IL-15 amino acid sequence.

SEQ ID NO:329 is a polypeptide encoding SEQ ID NO:317 to tether IL-15.

SEQ ID NO:330 is a nucleic acid sequence encoding GFP.

Detailed Description

I. Introduction to the invention

Adoptive cell therapy with TIL is an effective method of causing tumor regression in a variety of cancers, including leukemia and melanoma. The use of adjuvants including immunostimulants has been found to enhance adoptive cell therapies and extend such therapies to other solid tumors. However, co-administration of immunomodulators such as cytokines (e.g., interleukins) can cause undesirable toxicity due to the high doses required. Thus, providing such adjuvants at the correct time and site appears to be critical to avoiding such undesirable effects.

Provided herein are compositions and methods for treating cancer using a modified TIL, wherein the modified TIL comprises one or more immunomodulatory agents (e.g., cytokines) associated with its cell surface. Immunomodulators associated with TIL provide local immunostimulatory effects, which may advantageously enhance TIL survival and/or anti-tumor activity in patient recipients. Accordingly, the compositions and methods disclosed herein provide effective cancer therapies.

II. Definition of

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

As used herein, the terms "co-administration," "administration in combination with … …," "administration in combination with … …," "simultaneously" and "contemporaneously" encompass administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, e.g., a plurality of TILs) to a subject such that both active pharmaceutical ingredients and/or metabolites thereof are present in the subject at the same time. Co-administration includes simultaneous administration as separate compositions, administration at different times as separate compositions, or administration as a composition in which more than two active pharmaceutical ingredients are present. Preferably, the administration is simultaneous in separate compositions and in the form of a composition in which both agents are present.

The term "in vivo" refers to an event that occurs in a subject.

The term "in vitro" refers to an event that occurs outside the body of a subject. In vitro assays encompass cell-based assays employing living or dead cells, as well as cell-free assays that do not employ intact cells.

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

The term "rapid amplification" refers to an increase in the number of antigen-specific TILs by at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) over a period of one week, more preferably by at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a period of one week, or most preferably by at least about 100-fold over a period of one week. Various rapid amplification protocols are described herein.

Herein, "tumor-infiltrating lymphocytes" or "TILs" refer to a population of cells that is initially obtained as white blood cells that have left the subject's blood stream and migrated into a tumor. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TIL includes both primary TIL and secondary TIL. "Primary TIL" is TIL obtained from a patient tissue sample as described herein (sometimes referred to as "fresh collection"), "secondary TIL" is any population of expanded or proliferated TIL cells as described herein, including, but not limited to, bulk TIL (bulk TIL) and expanded TIL ("REP TIL" or "post-REP TIL"). The population of TIL cells may comprise genetically modified TIL.

"Population of cells" (including TIL) as used herein refers to a number of cells that have a common trait. Typically, the number of populations is in the range of 1 x 10 ⁶ to 1 x 10 ¹⁰, wherein different TIL populations comprise different numbers. For example, initial growth of primary TIL in the presence of IL-2 results in a population of bulk TIL of about 1X 10 ⁸ cells. REP expansion is typically performed to provide 1.5 x 10 ⁹ to 1.5 x 10 ¹⁰ cell populations for infusion.

By "cryopreserved TIL" is meant herein that the TIL, whether primary, bulk or amplified (REP TIL), is processed and stored in a range of about-150℃to-60 ℃. General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, "cryopreserved TIL" can be distinguished from frozen tissue samples that can be used as a source of primary TIL.

"Thawed cryopreserved TIL" herein refers to a population of TILs that have been previously cryopreserved and subsequently treated to return to room temperature or higher (including but not limited to cell culture temperatures or temperatures at which TILs may be administered to a patient).

TIL can generally be defined using biochemistry (using cell surface markers) or functionality (based on its ability to infiltrate tumors and effect treatment). TIL can generally be categorized by the expression of one or more of the following biomarkers: CD4, CD8, tcrαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1 and CD25. Additionally and alternatively, TIL may be functionally defined by the ability to infiltrate a solid tumor after reintroduction into a patient.

The term "cryopreservation medium" or "cryopreservation medium" refers to any medium that can be used to cryopreserve cells. Such media may include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, hypoThermosol, and combinations thereof. The term "CS10" refers to a cryopreservation medium obtained from Stemcell Technologies or Biolife Solutions. CS10 Medium is available under the trade name'CS10 "is referred to. The CS10 medium is a serum-free, animal-component-free medium comprising DMSO.

The term "central memory T cell" refers to a subset of T cells that are cd45r0+ and constitutively express CCR7 (CCR 7 ^hi) and CD62L (CD 62 ^hi) in humans. The surface phenotype of the central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2 and BMI1. The central memory T cells mainly secrete IL-2 and CD40L as effector molecules after TCR priming. Central memory T cells are predominantly present in the CD4 compartment of the blood, proportionally enriched in lymph nodes and tonsils in humans.

The term "effector memory T cells" refers to a subset of human or mammalian T cells, e.g., central memory T cells, that are cd45r0+, but have lost constitutive expression of CCR7 (CCR 7 ^lo) and are heterogeneous or low for CD62L expression (CD 62L ^lo). The surface phenotype of the central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. The transcription factors of central memory T cells include BLIMP a 1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines including interferon-gamma, IL 4-and IL-5 following antigen stimulation. Effector memory T cells are predominantly present in the CD8 compartment of blood, proportionally enriched in the lung, liver and intestinal tract in humans. Cd8+ effector memory T cells carry a large number of perforins.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for use in cell culture methods may be used in the methods of the invention. The containment system includes, for example (but not limited to), a containment G-vessel. Once the tumor fragments are added to the closed system, the system is not open to the external environment until the TIL is ready for administration to the patient.

As used herein, the terms "crush," "fragments," and "crushed" describe the process of destroying a tumor, including mechanical crushing methods, such as crushing, slicing, segmenting, and comminuting tumor tissue, as well as any other method for destroying the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells having rounded nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen presenting cells (PBMCs are one type of antigen presenting cells), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells that expand from peripheral blood. In some embodiments, the PBLs are isolated from whole blood or apheresis (apheresis) products from the donor. In some embodiments, the PBLs are isolated from whole blood or apheresis products from the donor by positively or negatively selecting T cell phenotypes (e.g., cd3+cd45+ T cell phenotypes).

The term "anti-CD 3 antibody" refers to an antibody or variant thereof, such as a monoclonal antibody, to the CD3 receptor in the T cell antigen receptor of mature T cells, and includes human, humanized, chimeric, murine, or mammalian antibodies. anti-CD 3 antibodies include OKT-3, also known as molumab. anti-CD 3 antibodies also include UHCT clones, also known as T3 and CD3 epsilon. Other anti-CD 3 antibodies include, for example, oxlizumab (oteliximab), telizumab (teplizuma), and velizumab (visilizumab).

The term "OKT-3" (also referred to herein as "OKT 3") refers to a monoclonal antibody to the CD3 receptor in the T cell antigen receptor of mature T cells, or a biological analogue or variant thereof, including human, humanized, chimeric or murine antibodies, including commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, santa Clarituximab Biotech, inc., miltenyi Biotech, inc., calif., U.S. and Moromomab or variant, conservative amino acid substitutions, glycoforms, or biological analogues thereof. The amino acid sequences of the heavy and light chains of Moromolizumab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO: 2). The hybridoma capable of producing OKT-3 was deposited with the American type culture Collection and assigned ATCC accession No. CRL 8001. Hybridomas capable of producing OKT-3 are also maintained in the European certified cell culture Collection (ECACC) and assigned accession number 86022706.

Table 1: amino acid sequence of Moromolizumab (exemplary OKT-3 antibodies)

The term "IL-2" (also referred to herein as "IL 2") refers to a T-cell growth factor known as interleukin-2, including all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs, and variants thereof. IL-2 is described, for example, in Nelson J.Immunol.) "2004,172,3983-88 and Malek, immunoannual review (Annu. Rev. Immunol.)" 2008,26,453-79, the disclosures of which are incorporated herein by reference. The amino acid sequences of recombinant human IL-2 suitable for use in the present invention are given in Table 2 (SEQ ID NO: SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as the form of aldesleukin (PROLEUKIN, available from multiple suppliers, 22 million IU per single use vial) and recombinant IL-2 supplied by CellGenix, inc. (CELLGRO GMP) of Mao Si, new hampshire, usa or ProSpec-Tany TechnoGene ltd. (catalog number CYT-209-b) of east allenix, new jersey, usa and other commercial equivalents from other suppliers. Albumin (Depropylamine acyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of about 15 kDa. The amino acid sequences of the aldesleukins suitable for use in the present invention are given in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including PEGylated IL2 prodrug Bei Peia interleukin (bempegaldesleukin) (NKTR-214, as shown in SEQ ID NO:4, PEGylated human recombinant IL-2 in which the average 6 lysine residues are N ⁶ substituted with [ (2, 7-bis { [ methyl poly (oxyethylene) ] carbamoyl } -9H-fluoren-9-yl) methoxy ] carbonyl), which are commercially available from Nektar Therapeutics of san francisco, california, U.S. or can be prepared by methods known in the art, such as those described in example 19 of International patent application publication No. WO 2018/132496 A1 or in example 1 of U.S. patent application publication No. US2019/0275133 A1, the disclosures of which are incorporated herein by reference. Bei Peia interleukin (NKTR-214) and other pegylated IL2 molecules suitable for use in the present invention are described in U.S. patent application publication No. US 2014/0328added 1 A1 and International patent application publication No. WO 2012/065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Pat. nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations suitable for use in the present invention are described in U.S. Pat. No.6,706,289, the disclosure of which is incorporated herein by reference.

In some embodiments, a suitable form of IL-2 for use in the present invention is THOR-707 available from Synthorx, inc. THOR-707 and the preparation and characterization of additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. patent application publication Nos. US2020/0181220 A1 and US2020/0330601A1, the disclosures of which are incorporated herein by reference. In some embodiments, the form of IL-2 suitable for use in the present invention is an interleukin-2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and binding the conjugate moiety of the isolated and purified IL-2 polypeptide at an amino acid position selected from the group consisting of: k35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72 and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. in some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments the amino acid positions are selected from E61, E62 and E68. In some embodiments, the amino acid position is at E62. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to a cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, the amino acid residue selected from the group consisting of K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargyloxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxy-phenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa (L-Dopa), fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-bromophenyl alanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-propyl-serine, O-tyrosine, O-L-methyl-tyrosine, O-L-tyrosine, O-phospho-tyrosine, O-L-4-phospho-tyrosine, O-L-tyrosine, O-phospho-L-tyrosine, P-phospho-serine, P-L-serine, P-phospho-L-phenylalanine, P-serine, P-O-L-phospho-serine, P-O-L-serine, P-O-L-4, L-phospho-serine, L-O-L-4, L, L-3- (2-naphthyl) alanine, 2-amino-3- ((2- ((3- (phenylmethoxy) -3-sidexypropyl) amino) ethyl) seleno-alkyl) propanoic acid, 2-amino-3- (phenylseleno-alkyl) propanoic acid or selenocysteine. In some embodiments, the IL-2 conjugate has reduced affinity for the IL-2 receptor alpha (IL-2Ralpha) subunit relative to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater than 99% reduction in binding affinity to IL-2rα relative to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or more relative to the wild-type IL-2 polypeptide. In some embodiments, the conjugate moiety attenuates or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugate moiety comprises a water-soluble polymer. In some embodiments, the additional conjugate moiety comprises a water-soluble polymer. In some embodiments, the water-soluble polymers each independently comprise polyethylene glycol (PEG), poly (propylene glycol) (PPG), a copolymer of ethylene glycol and propylene glycol, a poly (oxyethylated polyol), a poly (enol), a poly (vinylpyrrolidone), a poly (hydroxyalkyl methacrylamide), a poly (hydroxyalkyl methacrylate), a poly (saccharide), a poly (α -hydroxy acid), a poly (vinyl alcohol), a polyphosphazene, a Polyoxazoline (POZ), a poly (N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymers each independently comprise PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, the water-soluble polymers each independently comprise a polysaccharide. In some embodiments, the polysaccharide comprises polydextrose, polysialic acid (PSA), hyaluronic Acid (HA), amylose, heparin, heparan Sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the water-soluble polymers each independently comprise a glycan. In some embodiments, the water-soluble polymers each independently comprise a polyamine. In some embodiments, the conjugate moiety comprises a protein. In some embodiments, the additional conjugate moiety comprises a protein. In some embodiments, the proteins each independently comprise albumin, transferrin (transferrin), or transthyretin (TRANSTHYRETIN). In some embodiments, the proteins each independently comprise an Fc portion. In some embodiments, the proteins each independently comprise an Fc portion of an IgG. In some embodiments, the conjugate moiety comprises a polypeptide. In some embodiments, the additional conjugate moiety comprises a polypeptide. In some embodiments, the polypeptides each independently comprise an XTEN peptide, a glycine-rich high amino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamine. In some embodiments, the conjugate moiety binds directly to an isolated and purified IL-2 polypeptide. In some embodiments, the conjugate moiety is indirectly bound to the isolated and purified IL-2 polypeptide through a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, homobifunctional linkers comprise Luo Mante reagent (Lomant's reagent) dithiobis (succinimidyl propionate) DSP, 3' -dithiobis (sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl suberate) (BS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfoDST), glycosylbis (succinimidyl succinic acid) Ethylene (EGS), disuccinimidyl glutarate (DSG), N carbonate, N '-disuccinimidyl ester (DSC), dimethyl Diimidinate (DMA), dimethyl pimelate (DMP), dimethyl Xin Erya (DMS), dimethyl-3, 3' -dithiodipropionamidyl ester (DTBP), 1, 4-bis (3 '- (2' -pyridyldithio) propionamido) butane (DPDPB), bis-maleimidohexane (BMH), aryl halide containing compounds (DFDNB) (e.g., 1, 5-difluoro-2, 4-dinitrobenzene or 1, 3-difluoro-4, 6-dinitrobenzene), 4 '-difluoro-3, 3' -dinitrophenyl sulfone (DFDNPS), bis- [ beta- (4-azidosalicylamido) ethyl ] disulfide (BASED), formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3 '-dimethylbenzidine, benzidine, α' -p-diaminobiphenyl, diiodo-p-xylene sulfonic acid, N '-ethylene-bis (iodoacetamide) or N, N' -hexamethylene-bis (iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl 3- (2-pyridyldithio) propionate (sPDP), N-succinimidyl long-chain 3- (2-pyridyldithio) propionate (LC-sPDP), N-succinimidyl long-chain 3- (2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene (sMPT), sulfosuccinimidyl-6- [ α -methyl- α - (2-pyridyldithio) toluimidyl ] hexanoate (sulfo-LC-sMPT), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide), m-succinimidyl (m-4-succinimidyl) benzoate (MBs) and (MBs-succinimidyl) phenylimidyl (MBs) 42-succinimidyl), succinimidyl (4-iodoacetyl) aminobenzoate (sulfo-sIAB), succinimidyl-4- (p-maleimidophenyl) butyrate (sMPB), sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (sulfo-sMPB), N- (gamma-Maleimidobutyloxy) Succinimidyl (GMBs), N- (gamma-maleimidobutyryloxy) sulfosuccinimidyl (sulfo-GMBs), 6- ((iodoacetyl) amino) caproic acid succinimidyl ester (sIAX), 6- [6- (((iodoacetyl) amino) caproyl) amino ] caproic acid succinimidyl ester (sIAXX), 4- (((iodoacetyl) amino) methyl) cyclohexane-1-carboxylic acid succinimidyl ester (sIAC), 6- (((((4-iodoacetyl) amino) methyl) cyclohexane-1-carbonyl) amino) caproic acid succinimidyl ester (sIACX), iodoacetic acid p-nitrophenyl ester (84), carbonyl) and (phenylhydrazine reactive with (N-phenylhydrazine) (5) sulfonyl) butanoic acid, for example, reactive with (N-phenylhydrazine) and (N-phenylhydrazine) reactive with (N-4-phosphonomethyl) butanoic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxy-hydrazide-8 (M ₂C₂ H), 3- (2-pyridyldithio) propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl- (4-azidosalicylamide caproate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2- (p-azidosalicylamide) ethyl-1, 3 '-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-hydroxysuccinimidyl-6- (4' -azido-2 '-nitrophenylamino) caproate (sANPAH), sulfosuccinimidyl-6- (4' -azido-62 '-amino) ethyl-1, 3' -dithiopropionate (3242), N-hydroxysuccinimidyl-4-azido benzoate (N-nitro-62 '-nitro) nitro-2' -nitro caproate (N-azido) ANs-2-nitro-2-azido caproate (N-2-nitro-2-azido) 2-nitro benzoate Sulfosuccinimidyl-2- (m-azido-nitrobenzoylamino) -ethyl-1, 3 '-dithiopropionate (sAND), N-succinimidyl-4 (4-azidophenyl) 1,3' -dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl) -1,3 '-dithiopropionate (sulfo-sADP), 4- (p-azidophenyl) butanoic acid sulfosuccinimidyl (sulfo-sAPB), 2- (7-azido-4-methylcoumarin-3-acetamide) ethyl-1, 3' -dithiopropionate sulfosuccinimidyl (sAED), 7-azido-4-methylcoumarin-3-acetic acid sulfosuccinimidyl (sulfo-sAMCA), p-nitrophenyl (ρNPDP), p-nitrophenyl-2-diazon-3, 3-trifluoropropionate (DTP), 1- (p-azidosalicylamido) -4- (iodoacetamido) butane (57), N- [4- (azidosamidyl) ethyl-1, 3 '-dithiopropionate (sulfo-sAMCA), 7-azido-4-methylcoumarin-3-azidosuccinimidyl), p-nitropyruvic acid p-nitrophenyl (p-NPDP), p-nitrosalicylamide (62H-2-azidosuccinimidyl) 2' - (2-azidosuccinimidyl) propyl) 2-azido-2-carboxamido-2- (2-azidosuccinyl) propyl) azide, 4- (p-azidosalicylamido) butylamine (AsBA) or p-Azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, phe-Lys, val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleimide group, optionally comprising a maleimidocaproyl (mc), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises para-aminobenzyl alcohol (PAB), para-aminobenzyloxycarbonyl (PABC), derivatives or analogs thereof. In some embodiments, the conjugate moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugate moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 forms suitable for use in the present invention are fragments of any of the IL-2 forms described herein. In some embodiments, the form of IL-2 suitable for use in the present invention is pegylated as disclosed in U.S. patent application publication No. US2020/0181220 A1 and U.S. patent application publication No. US2020/0330601 A1. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises a sequence that hybridizes to SEQ ID NO:5 an amino acid sequence having at least 80% sequence identity; reference is made to SEQ ID NO:5 for amino acid position AzK at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence relative to SEQ ID NO:5, the N-terminus of one residue is deleted. In some embodiments, the IL-2 forms suitable for use in the present invention lack IL-2Rα linkage, but remain normally bound to the intermediate affinity IL-2Rβ - γ signaling complex. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises a sequence that hybridizes to SEQ ID NO:5 an amino acid sequence having at least 90% sequence identity; and reference to SEQ ID NO:5 for amino acid position AzK at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises a sequence that hybridizes to SEQ ID NO:5 having at least 95% sequence identity; reference is made to SEQ ID NO:5 for amino acid position AzK at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) covalently linked to a conjugate moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises a sequence that hybridizes to SEQ ID NO:5 an amino acid sequence having at least 98% sequence identity; reference is made to SEQ ID NO:5, for amino acid substitutions AzK at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72.

In some embodiments, the form of IL-2 suitable for use in the present invention is netile Jin (also known as ALKS-4230 (SEQ ID NO: 6), which is available from Alkermes, inc.). Nefion Jin, also known as a human interleukin 2 fragment (1-59) variant (Cys ¹²⁵>Ser⁵¹), fused to a human interleukin 2 fragment (62-132) by a peptidyl linker (⁶⁰GG⁶¹), fused to a human interleukin 2 receptor alpha chain fragment (139-303) by a peptidyl linker (¹³³GSGGGS¹³⁸), produced in Chinese Hamster Ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133) -peptide [ Cys ¹²⁵ (51) > Ser ] -mutant (1-59) fused to human interleukin 2 (IL-2) (4-74) -peptide (62-132) via a G ₂ peptide linker (60-61) and to human interleukin 2 receptor alpha chain (IL 2R subunit alpha, IL2R alpha, IL2 RA) (1-165) -peptide (139-303) via a GSG ₃ S peptide linker (133-138), produced in Chinese Hamster Ovary (CHO) cells. The amino acid sequence of nefiv Jin is set forth in SEQ ID NO: 6. In some embodiments, netile Jin has the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbering in SEQ ID NO: 6), and a glycosylation site at the following positions: n187, N206, T212 (using the numbering in SEQ ID NO: 6). The preparation and properties of nefiv Jin and other alternative forms of IL-2 suitable for use in the present invention are described in U.S. patent application publication No. US 2021/0038684A1 and U.S. patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, the form of IL-2 suitable for use in the present invention is as set forth in SEQ ID NO:6, a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity. In some embodiments, the IL-2 forms suitable for use in the present invention have the amino acid sequence of SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, the form of IL-2 suitable for use in the present invention is a polypeptide comprising SEQ ID NO:7 or a variant, variant or derivative thereof. In some embodiments, the form of IL-2 suitable for use in the present invention is a polypeptide comprising a sequence corresponding to SEQ ID NO:7 or a variant, fragment or derivative thereof having an amino acid sequence of at least 80%, at least 90%, at least 95% or at least 90% sequence identity. Other forms of IL-2 suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference. Alternatively, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1rα or a protein having at least 98% amino acid sequence identity to IL-1rα and receptor antagonist activity of IL-rα, the second fusion partner comprises an immunoglobulin comprising in whole or in part an Fc region, wherein the mucin domain polypeptide linker comprises the amino acid sequence of SEQ ID NO:8 or with SEQ ID NO:8, the half-life of the fusion protein is improved compared to the fusion of the first fusion partner to the second fusion partner without the mucin domain polypeptide linker.

Table 2: the amino acid sequence of interleukin.

In some embodiments, the form of IL-2 suitable for use in the present invention includes an antibody cytokine transplantation protein comprising: a heavy chain variable region (V _H) comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (V _L) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof, which is grafted into the CDRs of V _H or V _L, wherein the antibody cytokine transplantation protein expands T effector cells in preference to regulatory T cells. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (V _H) comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (V _L) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into the CDRs of V _H or V _L, wherein the IL-2 molecule is a mutein and the antibody cytokine transplantation protein expands T effector cells in preference to regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody described in U.S. patent application publication No. US2020/0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine graft protein comprises: a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into the CDRs of V _H or V _L, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine transplantation protein expands T effector cells in preference to regulatory T cells, the antibody further comprising an IgG class heavy chain and an IgG class light chain selected from the group consisting of: comprising SEQ ID NO:39 and an IgG class light chain comprising SEQ ID NO:38, an IgG class heavy chain; comprising SEQ ID NO:37 and an IgG class light chain comprising SEQ ID NO:29 IgG class heavy chain; comprising SEQ ID NO:39 and an IgG class light chain comprising SEQ ID NO:29 IgG class heavy chain; an IgG class light chain comprising SEQ ID NO 37 and an amino acid sequence comprising SEQ ID NO: 38.

In some embodiments, the IL-2 molecule or fragment thereof is grafted into HCDR1 of V _H, wherein the IL-2 molecule is a mutein. In some embodiments, the IL-2 molecule or fragment thereof is grafted into HCDR2 of V _H, wherein the IL-2 molecule is a mutein. In some embodiments, the IL-2 molecule or fragment thereof is grafted into HCDR3 of V _H, wherein the IL-2 molecule is a mutein. In some embodiments, the IL-2 molecule or fragment thereof is grafted into LCDR1 of V _L, wherein the IL-2 molecule is a mutein. In some embodiments, the IL-2 molecule or fragment thereof is grafted into LCDR2 of V _L, wherein the IL-2 molecule is a mutein. In some embodiments, the IL-2 molecule or fragment thereof is grafted into LCDR3 of V _L, wherein the IL-2 molecule is a mutein.

The insertion of the IL-2 molecule may be at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDRs, wherein the IL2 sequence does not frame the CDR sequences. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL-2 sequence replaces all or a portion of the CDR sequence. IL-2 molecular substitutions may be at the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. IL-2 molecular substitutions may be as few as one or two amino acids of the CDR sequence or the entire CDR sequence.

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

In some embodiments, the IL-2 molecules described herein are IL-2 muteins. In some cases, the IL-2 mutein comprises the R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15. in some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. patent application publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference.

In some embodiments, the antibody cytokine graft protein comprises a sequence selected from the group consisting of SEQ ID NOs: 16. SEQ ID NO: 19. SEQ ID NO:22 and HCDR1 of SEQ ID NO 25. In some embodiments, the antibody cytokine graft protein comprises a sequence selected from the group consisting of SEQ ID NOs: 7. SEQ ID NO: 10. SEQ ID NO:13 and SEQ ID NO:16 HCDR1. In some embodiments, the antibody cytokine graft protein comprises HCDR1 selected from the group consisting of: selected from SEQ ID NOs: 17. SEQ ID NO: 20. SEQ ID NO:23 and SEQ ID NO: HCDR2 of 26. In some embodiments, the antibody cytokine graft protein comprises a sequence selected from the group consisting of SEQ ID NOs: 18. SEQ ID NO: 21. SEQ ID NO:24 and SEQ ID NO: HCDR3 of 27. In some embodiments, the antibody cytokine graft protein comprises a V _H region comprising the amino acid sequence of SEQ ID NO:28, and a sequence of amino acids. In some embodiments, the antibody cytokine graft protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine graft protein comprises a V _L region comprising the amino acid sequence of SEQ ID NO:36, and a nucleotide sequence of 36. In some embodiments, the antibody cytokine graft protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:37, and a sequence of amino acids thereof. In some embodiments, the antibody cytokine graft protein comprises a V _H region comprising the amino acid sequence of SEQ ID NO:28, an amino acid sequence of seq id no; and a V _L region comprising SEQ ID NO:36, and a nucleotide sequence of 36. In some embodiments, the antibody cytokine graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO 29; and a light chain region comprising SEQ ID NO:37, and a sequence of amino acids thereof. In some embodiments, the antibody cytokine graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29, an amino acid sequence of seq id no; and a light chain region comprising SEQ ID NO:39, and a sequence of amino acids. In some embodiments, the antibody cytokine graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38, an amino acid sequence of seq id no; and a light chain region comprising SEQ ID NO:37, and a sequence of amino acids thereof. In some embodiments, the antibody cytokine graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38, a base acid sequence of seq id no; and a light chain region comprising SEQ ID NO:39, and a sequence of amino acids. In some embodiments, the antibody cytokine graft protein comprises an igg.il2f71a.h1 or igg.il2r67a.h1 or variant, derivative or fragment thereof, or a conservative amino acid substitution thereof, or a protein having at least 80%, at least 90%, at least 95% or at least 98% sequence identity thereto, of U.S. patent application publication No. 2020/0270334 A1. In some embodiments, the antibody component of the antibody cytokine transplantation proteins described herein comprises an immunoglobulin sequence, framework sequence, or CDR sequence of palivizumab. In some embodiments, the serum half-life of the antibody cytokine transplantation proteins described herein is longer than that of a wild-type IL-2 molecule (e.g., without limitation, aldesleukin or a comparable molecule). In some embodiments, the antibody cytokine transplantation proteins described herein have the sequences as set forth in table 3.

Table 3: exemplary palivizumab antibody-IL-2 graft protein sequences

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The term "IL-4" (also referred to herein as "IL 4") refers to a cytokine called interleukin 4, which is produced by Th 2T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th 0 cells) into Th 2T cells. Steinke and Borish, respiratory research (respir.res.) 2001,2,66-70. After activation by IL-4, th 2T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is available from a number of suppliers including ProSpec-Tany TechnoGene Ltd (catalog number CYT-211) of Dongtoron Rake, new Jersey, U.S. and Simer Feishmania science and technology company (ThermoFisher Scientific, inc.) of Woltherm, massachusetts, U.S. and Gibco CTP0043 (human IL-15 recombinant protein). The amino acid sequences of recombinant human IL-4 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 9).

The term "IL-7" (also referred to herein as "IL 7") refers to glycosylated tissue-derived cytokines known as interleukin 7, which are obtainable from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, blood 2002, 99, 3892-904.IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and a common gamma chain receptor, which belongs to a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the present invention is available from a number of suppliers including ProSpec-Tany TechnoGene Ltd (catalog number CYT-254) of Dongtoron Rake, new Jersey, U.S. and Siemens Feishan technologies, inc. (human IL-15 recombinant protein, catalog number Gibco PHC 0071) of Walsh, massachusetts, U.S. The amino acid sequences of recombinant human IL-7 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 10).

The term "IL-15" (also referred to herein as "IL-15") refers to a T-cell growth factor known as interleukin-15, including all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiouri, blood 2001,97,14-32, the disclosures of which are incorporated herein by reference. IL-15 shares β and γ signal receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain of molecular mass 12.8kDa containing 114 amino acids (and an N-terminal methionine). Recombinant human IL-15 is available from a number of suppliers including ProSpec-Tany TechnoGene Ltd (catalog number CYT-230-b) of Dongtorsemide, N.J., U.S. and Siemens Feishan technologies, inc. (human IL-15 recombinant protein, catalog number 34-8159-82) of Wolsephm, massachusetts, U.S. The amino acid sequences of recombinant human IL-15 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 11).

The term "IL-21" (also referred to herein as "IL 21") refers to pleiotropic cytokine proteins known as interleukin-21, including all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, nature reviews: drug discovery (Nat. Rev. Drug. Disc.) 2014,13,379-95, the disclosure of which is incorporated herein by reference. IL-21 is produced primarily by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain of 132 amino acids having a molecular mass of 15.4 kDa. Recombinant human IL-21 is available from a number of suppliers including ProSpec-Tany TechnoGene Ltd (catalog number CYT-408-b) of Dongtorsemide, N.J., U.S. and Siemens Feishan technologies, inc. (human IL-21 recombinant protein, catalog number 14-8219-80) of Woltherm, massachusetts, U.S. The amino acid sequences of recombinant human IL-21 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 12).

When an "anti-tumor effective amount", "tumor inhibiting effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician considering the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It is generally stated that the pharmaceutical compositions described herein comprising tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) can be administered at doses of 10 ⁴ to 10 ¹¹ cells/kg body weight (e.g., 10 ⁵ to 10 ⁶、10⁵ to 10 ¹⁰、10⁵ to 10 ¹¹、10⁶ to 10 ¹⁰、10⁶ to 10 ¹¹、10⁷ to 10 ¹¹、10⁷ to 10 ¹⁰、10⁸ to 10 ¹¹、10⁸ to 10 ¹⁰、10⁹ to 10 ¹¹ or 10 ⁹ to 10 ¹⁰ cells/kg body weight), including all integer values within those ranges. TIL (in some cases including genetically modified cytotoxic lymphocytes) compositions may also be administered at these doses multiple times. TIL (including in some cases genetically engineered TIL) can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., rosenberg et al, journal of New england medicine (New eng.j. Of med.)) 1988,319,1676. The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient's disease condition and adjusting the treatment accordingly.

The terms "hematopoietic malignancy", "hematopoietic malignancy" or related terms refer to cancers and tumors of mammalian hematopoietic and lymphoid tissues (including, but not limited to, blood, bone marrow, lymph node, and lymphoid tissue). Hematopoietic malignancies are also known as "liquid tumors". Hematopoietic malignancies include, but are not limited to, acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Lymphoma (CLL), small Lymphocytic Lymphoma (SLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), hodgkin's lymphoma, and non-hodgkin's lymphoma. The term "B cell hematological malignancy" refers to a malignancy of the hematopoietic system that affects B cells.

The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematopoietic malignancies. TIL obtained from liquid tumors may also be referred to herein as bone Marrow Infiltrating Lymphocytes (MILs). TIL obtained from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBL. The terms MILs, TIL and PBL are used interchangeably herein and differ based solely on the tissue type of the derived cells.

As used herein, the term "microenvironment" may refer to a solid or hematological tumor microenvironment as a whole or may refer to a subset of individual cells within the microenvironment. As used herein, tumor microenvironment refers to the following complex mixture: cells, soluble factors, signaling molecules, extracellular matrix and mechanical signals that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, encourage therapeutic resistance and provide an ecolocus for dominant metastasis growth, as described in Swartz et al, cancer research (Cancer res.), 2012,72,2473. Although tumors express antigens that should be recognized by T cells, it is rare that the immune system clears the tumor due to immunosuppression of the microenvironment.

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

Experimental findings indicate that lymphocyte depletion plays a key role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system ("cytokine repertoire") prior to adoptive transfer of tumor-specific T lymphocytes. Thus, some embodiments of the invention employ a lymphocyte depletion step (sometimes also referred to as "immunosuppressive modulation") in the patient prior to introducing the TIL of the invention.

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

The terms "treat," "treating," and similar terms refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects attributable to the disease. As used herein, "treating" encompasses any treatment of a disease in a mammal, particularly a human, including: (a) Preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., suppressing its development or progression; and (c) alleviating the disease, i.e., causing regression of the disease and/or alleviating one or more symptoms of the disease. "treating" is also intended to encompass delivering an agent so as to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treating" encompasses the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

When used with reference to a portion of a nucleic acid or protein, the term "heterologous" means that the nucleic acid or protein comprises two or more subsequences that are found in nature not in the same relationship to each other. For example, nucleic acids are typically produced recombinantly, having more than two sequences from unrelated genes arranged to make new functional nucleic acid sequences, such as a promoter from one source and a coding region from another source or a coding region from a different source. Similarly, a heterologous protein means that the protein comprises more than two subsequences that are not found in the same relationship to each other in nature (e.g., fusion proteins).

In the context of two or more nucleic acids or polypeptides, the terms "sequence identity", "percent identity", and "percent sequence identity" (or synonyms thereof, e.g., "99% identity") refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide or amino acid residues that are the same, when compared and aligned (optionally introducing gaps) to achieve maximum correspondence, and do not consider any conservative amino acid substitutions as part of sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain an alignment of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, BLAST program packages available from the national center for Biotechnology information BLAST website of the United states government. The comparison between two sequences can be made using the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genntech), or MegAlign (available from DNASTAR), are additional software programs available to the general public for aligning sequences. One skilled in the art can determine appropriate parameters for maximum alignment by specific alignment software. In some embodiments, default parameters of the alignment software are used.

As used herein, the term "variant" encompasses, but is not limited to, antibodies or fusion proteins comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by more than one substitution, deletion, and/or addition at certain positions within or adjacent to the amino acid sequence of the reference antibody. Variants may contain more than one conservative substitution in their amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, for example, substitutions like charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

Herein, "tumor-infiltrating lymphocytes" or "TILs" refer to a population of cells that is initially obtained as white blood cells that have left the subject's blood stream and migrated into a tumor. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TIL includes both primary TIL and secondary TIL. "Primary TIL" is a cell obtained from a patient tissue sample as described herein (sometimes referred to as "freshly harvested"), "secondary TIL" is any population of expanded or proliferated TIL cells as described herein, including, but not limited to, the subject TILs and expanded TILs ("REP TILs") and "reREP TIL") described herein. reREP TIL may include, for example, a second amplified TIL or a second additional amplified TIL (e.g., TIL described in step D of fig. 8, including TIL referred to as reREP TIL).

TIL can generally be defined biochemically (using cell surface markers) or functionally (based on its ability to infiltrate tumors and effect treatment). TIL can be classified generally by expressing more than one of the following biomarkers: CD4, CD8, tcrαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1 and CD25. Additionally and alternatively, TIL may be functionally defined by its ability to infiltrate solid tumors after reintroduction into a patient. TIL may be further characterized by potency-for example, if, for example, interferon (IFN) release is greater than about 50pg/mL, greater than about 100pg/mL, greater than about 150pg/mL, or greater than about 200pg/mL, then TIL may be considered potent. TIL may be considered potent if, for example, interferon (ifnγ) release is greater than about 50pg/mL, greater than about 100pg/mL, greater than about 150pg/mL, or greater than about 200pg/mL, greater than about 300pg/mL, greater than about 400pg/mL, greater than about 500pg/mL, greater than about 600pg/mL, greater than about 700pg/mL, greater than about 800pg/mL, greater than about 900pg/mL, greater than about 1000 pg/mL.

The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include altering the linkage between sugar moieties, base moieties and/or deoxyribonucleotides in the oligonucleotide.

The term "RNA" defines a molecule comprising at least one ribonucleotide residue. A "ribonucleotide" is defined as a nucleotide that has a hydroxy group at the 2' -position of the b-D-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA), and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of more than one nucleotide. The nucleotides in the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, may also be incorporated into the described compositions and methods.

The term "about" or "approximately" refers to a statistically significant range of values. This range may be within an order of magnitude of the given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, even more preferably within 5%. The allowable differences encompassed by the term "about" or "approximately" depend on the particular system under study, and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" refer to dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics that are not and need not be exact, but may be approximated and/or greater or lesser, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, as well as other factors known to those of skill in the art. Generally, a dimension, size, formulation, parameter, shape, or other quantity or feature is "about" or "about" whether or not so explicitly stated. It should be noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangement.

When used in the appended claims, the transitional terms "comprising," "consisting essentially of …," and "consisting of …," when used in their original and modified forms, are intended to define the scope of the claims relative to additional claim elements or steps (if present) not recited. The term "comprising" is intended to be inclusive or open ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of …" does not include any element, step or material other than the one specified in the claims, in which case impurities generally associated with the specified material are excluded. The term "consisting essentially of …" limits the scope of the claims to elements, steps, or materials specified, elements, steps, or materials that do not substantially affect the basis and novel features of the claimed invention. In alternative embodiments, all compositions, methods, and kits embodying the invention described herein may be more specifically defined by any transitional term "comprising," consisting essentially of …, "and" consisting of ….

The term "antibody (antibodies)" and its plural form "antibodies" refer to intact immunoglobulins as well as any antigen binding fragment ("antigen binding portion") or single chain thereof. "antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V _H) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (abbreviated herein as V _L) and a light chain constant region. The light chain constant region is composed of one domain C _L. The V _H and V _L regions of antibodies can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs), which can be interspersed with regions that are more conserved, termed Framework Regions (FR). Each V _H and V _L consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with more than one epitope. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of binding to an antibody or TCR if presented by a Major Histocompatibility Complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. The antigen is additionally capable of being recognized by the immune system. In some embodiments, the antigen is capable of inducing a humoral or cellular immune response that activates B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contain or be linked to a Th cell epitope. An antigen may also have more than one epitope (e.g., B epitope and T epitope). In some embodiments, the antigen will preferably react with its corresponding antibody or TCR, typically in a highly specific and selective manner, but not with a variety of other antibodies or TCRs that may be induced by other antigens.

The terms "monoclonal antibody", "mAb", "monoclonal antibody composition" or a plurality thereof refer to a preparation of antibody molecules of a single molecule composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for certain receptors can be made using knowledge and techniques in the following arts: i.e., injecting the test subject with the appropriate antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell that does not otherwise produce immunoglobulins, such as an escherichia coli cell, simian COS cell, chinese Hamster Ovary (CHO) cell, or myeloma cell, to achieve monoclonal antibody synthesis in the recombinant host cell. Recombinant production of antibodies will be described in more detail below.

As used herein, the term "antigen binding portion" or "antigen binding fragment" of an antibody (or, in brief, an "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of V _L、V_H、C_L and CH1 domains; (ii) A F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) An Fd fragment consisting of V _H and a CH1 domain; (iv) Fv fragments consisting of the V _L and V _H domains of the single arm of the antibody; (v) Domain antibodies (dAb) fragments (Ward et al, nature, 1989,341,544-546) which may consist of one V _H or one V _L domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment (V _L and V _H) are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that can make the two domains into a single protein chain (known as a single chain Fv (scFv)) that pairs V _L with the V _H region to form a monovalent molecule; see, e.g., bird et al, science 1988,242,423-426; huston et al, proc. Natl. Acad. Sci. USA, 1988,85,5879-5883. Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments used are screened in the same manner as the whole antibody. In some embodiments, the scFv protein domain comprises a V _H portion and a V _L portion. The scFv molecule is denoted V _L-L-V_H in the case where the V _L domain is the N-terminal portion of the scFv molecule, or V _H-L-V_L in the case where the V _H domain is the N-terminal portion of the scFv molecule; U.S. Pat. nos. 4,946,778; raag and M.Whitlow, single Chain Fvs, [ American society of laboratory Biotechnology (FASEB) ], volume 9:73-80 (1995); and R.E.bird and B.W.Walker, single Chain Antibody Variable Regions, trends In Biotechnology (TIBTECH), volume 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

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

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

As used herein, the term "recombinant human antibody" includes all human antibodies produced, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for human immunoglobulin genes or hybridomas (described further below) prepared therefrom; (b) Isolated antibodies from host cells transformed to express human antibodies, e.g., from a hybridoma; (c) antibodies isolated from a recombinant, combinatorial human antibody library; and (d) antibodies produced, expressed, produced or isolated by any other means that involves splicing the human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies have framework and CDR regions derived from the variable regions of human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subject to in vitro mutations (or in vivo somatic mutations when transgenic animals of human Ig sequences are used), so the amino acid sequences of the V _H and V _L regions of the recombinant antibodies are sequences that, while derived from and associated with human germline V _H and V _L sequences, may not naturally occur in vivo within the human antibody germline.

As used herein, "isotype (isotype)" refers to the class of antibodies (e.g., igM or IgG 1) encoded by the heavy chain constant region gene.

The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

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

The terms "humanized antibody", "humanized antibody" and "humanized" refer to antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences. Other framework region modifications may be made in the human framework sequence. Humanized versions of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from 15 hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and capacity. In some cases, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications were made to further optimize antibody efficacy. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. Alternatively, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, nature 1986,321,522-525; riechmann et al Nature 1988,332,323-329; and Presta, new structure biology (curr.op.struct.biol.) 1992,2,593-596. The antibodies described herein may also be modified to use any Fc variant known to confer effector function and/or improved (e.g., reduced) FcR binding. Fc variants may include any of the amino acid substitutions disclosed below: international patent application publication Nos. WO 1988/07089A1, WO 1996/14339A1, WO 1998/05787A1, WO 1998/23289A1, WO 1999/51642A1, WO 99/58552 A1, WO 2000/09560A2, WO 2000/32767A1, WO 2000/42072A2, WO 2002/44215A2, WO 2002/060919 A2, WO 2003/074569 A2, WO 2004/016750A2, WO 2004/029207 A2, WO 2004/035752 A2, WO 2004/351 A2, WO 2004/074455 A2, WO 2004/099249 A2, WO 2005/0632217 A2, WO 2005/963 A1, WO 2005/077981 A2, WO 2005/01625 A2, WO 2006/029207 A2, WO 2006/03505WO 2006/035752 A2, WO 2004/07005WO WO 2004/351 A2, WO 2004/0907055 A2, WO 2005/0901945 A2; U.S. Pat. nos. 5,648,260; 5,739,277; no. 5,834,250; no. 5,869,046; 6,096,871 th sheet; 6,121,022; 6,194,551; 6,242,195 th sheet; 6,277,375; 6,528,624 th sheet; 6,538,124 th sheet; no. 6,737,056; 6,821,505 th sheet; 6,998,253 th sheet; 7,083,784 th sheet; the disclosure of which is incorporated herein by reference.

The term "chimeric antibody" means an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

A "bifunctional antibody" is a small antibody fragment having two antigen binding sites. The fragment comprises a heavy chain variable domain (V _H) linked to a light chain variable domain (V _L) in the same polypeptide chain (V _H-V_L or V _L-V_H). By using a linker that is too short to allow pairing between two domain structures on the same strand, the domains are forced to pair with the complementary domain of the other strand, creating two antigen binding sites. Bifunctional antibodies are described in more detail in, for example, european patent EP 404,097, international patent publication No. WO 93/11161; bolliger et al, proc. Natl. Acad. Sci. USA 1993,90,6444-6448.

The term "glycosylation" refers to a modified derivative of an antibody. Non-glycosylated antibodies lack glycosylation. Glycosylation can be altered, for example, to increase the affinity of antibodies for antigens. Such carbohydrate modification may be accomplished, for example, by altering more than one glycosylation site within the antibody sequence. For example, more than one amino acid substitution may be made to eliminate more than one variable region framework glycosylation site, thereby eliminating glycosylation at that site. Deglycosylation can increase the affinity of the antibody for the antigen, as described in U.S. Pat. nos. 5,714,350 and 6,350,861. Additionally or alternatively, antibodies with altered glycosylation patterns may be produced, such as low fucosylation antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structure. Such altered glycosylation patterns have been shown to increase the ability of antibodies. Such carbohydrate modification may be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing the recombinant antibodies of the invention to thereby produce antibodies with altered glycosylation. For example, cell lines Ms704, ms705 and Ms709 lack the fucosyltransferase gene, FUT8 (α (1, 6) fucosyltransferase), such that antibodies expressed in the Ms704, ms705 and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, ms705 and Ms709 FUT 8-/-cell lines were formed by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. patent publication No. 2004/010704 or Yamane-Ohnuki et al, biotechnology and bioengineering (Biotechnol. Bioeng.), 2004,87,614-622). As another example, european patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such a cell line exhibit low fucosylation by reducing or eliminating an α1,6 linkage-associated enzyme, as well as the following cell lines: with low or no enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of the antibody, e.g., the cell line is the rat myeloma cell line YB2/0 (ATCC CRL 1662). International patent publication WO 03/035835 describes variant CHO cell lines, lec 13 cells, which have a reduced capacity to attach fucose to Asn (297) linked carbohydrates, also resulting in low fucosylation of antibodies expressed in host cells (see also Shields et al, journal of biochemistry (j. Biol. Chem.)) 2002,277,26733-26740 international patent publication WO 99/54342 describes cell lines engineered to express glycoprotein modified glycosyltransferases (e.g., β (1, 4) -N-acetylglucosamine transferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisected GlcNac structure, thereby enhancing ADCC activity of antibodies (see also uma et al, natural. Biotechnology (nat. Biotech.)) 1999,17,176-180. Alternatively fucosidase may be used to untangling fucose residues of antibodies, e.g., fucosidase- α -3779 from fucosidase, such as described in bio-35, etc.).

"PEGylation" refers to a modified antibody or fragment thereof that is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which more than one PEG group is attached to the antibody or antibody fragment. For example, pegylation can increase the biological (e.g., serum) half-life of an antibody. Preferably, the pegylation is performed by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derive other proteins, such as mono (C ₁-C₁₀) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated is a deglycosylated antibody. The pegylation methods are known in the art and can be applied to the antibodies of the present invention, for example as described in european patent No. EP 0154316 and EP 0401384 and us patent No. 5,824,778, the respective disclosures of which are incorporated herein by reference.

The term "biological analog" refers to the following biological products (including monoclonal antibodies or proteins): despite minor differences in clinically inactive ingredients, there are no clinically significant differences in safety, purity and efficacy of the product between this biological product and the reference product, which are highly similar to the us licensed reference biological product. Furthermore, a similar biological or "biological analog" drug is a biological drug that is similar to another biological drug that has been authorized for use by the european drug administration. The term "biological analogue" is also synonymously used by other national and regional regulatory authorities. A biologic product or biologic drug is a drug made or derived from a biologic source (e.g., bacteria or yeast). It may consist of relatively small molecules (e.g. human insulin or erythropoietin) or complex molecules (e.g. monoclonal antibodies). For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), the protein that is approved by the pharmaceutical regulatory agency as reference aldesleukin is "aldesleukin bioanalog" or "aldesleukin bioanalog". In europe, a similar biological or "biological analog" drug is a biological drug that is similar to another biological drug that has been authorized for use by the european drug administration (EMA). The relevant legal basis for European similar biological applications is code (EC) No. 726/2004 No. 6 and instruction 2001/83/EC No. 10 (4), revised and thus in Europe, biological analogs are the subject of an authorized or granted application that can be granted, approved, according to code (EC) No. 726/2004 No. 6 and instruction 2001/83/E No. 10 (4). The original biopharmaceutical product that is authorized may be referred to as a "reference drug" in europe. Some requirements of the product as a biosimilar are summarized in the guidelines for similar biopharmaceutical products. In addition, product specific guidelines, including guidelines associated with monoclonal antibody biological analogs, are provided by EMA on a product-by-product basis and posted on their web sites. The biological analogs as described herein may be similar to a reference drug in terms of quality characteristics, biological activity, mechanism of action, safety profile, and/or efficacy. In addition, the biological analogs can be used or intended to be used to treat the same condition as the reference drug. Thus, a biological analog as described herein may be considered to have similar or highly similar quality characteristics to a reference drug. Alternatively or additionally, a biological analog as described herein may be considered to have similar or highly similar biological activity to a reference drug. Alternatively or additionally, the biological analogs as described herein may be considered to have a similar or highly similar safety profile as the reference drug. Alternatively or additionally, the biological analogs as described herein may be considered to have similar or highly similar efficacy to the reference drug. As described herein, in europe, biological analogs have been compared to reference drugs authorized by EMA. However, in some cases, in some studies, the biological analogs may be compared to biopharmaceuticals that are authorized outside of the european economy (non-EEA authorized "comparator"). Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biological analog" also relates to biopharmaceuticals that have been compared to or may be compared to a non-EEA authorized comparison. Some biological analogs are proteins, such as antibodies, antibody fragments (e.g., antigen binding portions), and fusion proteins. A protein biological analog may have an amino acid sequence with minor modifications in the amino acid structure (including, for example, deletions, additions and/or substitutions of amino acids) that do not significantly affect the function of the polypeptide. A biological analog may comprise an amino acid sequence that has 97% or more (e.g., 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of its reference drug. The biological analogs may comprise more than one post-translational modification, such as, but not limited to, glycosylation, oxidation, deamidation, and/or truncation, that is different from the post-translational modification of the reference drug, provided that these differences do not result in a change in the safety and/or efficacy of the drug. The biological analog may have the same or a different glycosylation pattern than the reference drug. In particular, although not exclusively, biological analogs may have different glycosylation patterns if the differences solve or are intended to solve the safety issues associated with the reference drug. In addition, the biological analogs may differ from the reference drug, for example, in their strength, pharmaceutical form, formulation, excipient, and/or manner of presentation, provided that the safety and efficacy of the drug are not compromised. Biological analogs may comprise differences in, for example, pharmacokinetic (PK) and/or Pharmacodynamic (PD) profiles as compared to a reference drug, but are still considered sufficiently similar to a reference drug to be ready for authorization or to be considered suitable for authorization. In some cases, the biological analogs exhibit different binding characteristics compared to a reference drug, wherein the regulatory body (e.g., EMA) does not treat these different binding characteristics as an obstacle to the authorization of a similar biological product. The term "biological analogue" is also synonymously used by other national and regional regulatory authorities.

III. immunomodulator-related tumor infiltrating lymphocytes

Provided herein are modified tumor-infiltrating lymphocytes (TILs) comprising one or more immunomodulatory agents that bind to the surface of TIL cells. In some embodiments, the modified TILs of the present invention exhibit enhanced in vivo survival, proliferation, and/or anti-tumor effects in patient recipients.

The immunomodulatory agents may be linked to the TILs disclosed herein (e.g., therapeutic TILs provided herein) using any suitable method. In some embodiments, the one or more immunomodulatory agents are part of an immunomodulatory fusion protein linked to the surface of a TIL cell. In some embodiments, the one or more immunomodulatory agents are included as part of a nanoparticle that binds to the surface of a TIL cell. The immunomodulator may be any immunomodulator that promotes TIL survival, proliferation and/or anti-tumor effects in the patient recipient. In some embodiments, the immunomodulator is a cytokine (e.g., interleukin). In exemplary embodiments, the TIL comprises IL-12, IL-15 and/or IL-21.

Any suitable population of TILs (including TILs prepared using the methods of manufacture described herein) can be modified to produce the compositions of the invention described herein. In some embodiments, the modified TIL is derived from a TIL produced during any step of the process 2A methods disclosed herein (see, e.g., fig. 2-6). In an exemplary embodiment, the modified TIL is derived from a TIL produced during any step of the GEN 3 method disclosed herein (see, e.g., fig. 7). In some embodiments, the TIL is a PD-1 positive TIL derived from the methods disclosed herein.

Aspects of the modified TILs of the invention are described in further detail herein.

A. Immunomodulatory fusion proteins

In some embodiments, the modified TILs provided herein comprise an immunomodulatory fusion protein comprising an immunomodulatory agent (e.g., a cytokine) linked to a moiety that facilitates tethering of the immunomodulatory agent to the surface of the TIL. In some embodiments, the fusion protein comprises a cell membrane anchor portion (transmembrane domain). In certain embodiments, the fusion protein comprises a TIL surface antigen binding moiety that binds to a TIL surface antigen. Aspects of these fusion proteins are discussed in further detail below.

1. Membrane anchored immunomodulatory fusion proteins

In some embodiments, the modified TIL provided herein comprises a membrane-anchored immunomodulatory fusion protein. The membrane anchored immunomodulatory fusion protein comprises one or more of an immunomodulatory agent (e.g., a cytokine) linked to a membrane anchor portion. In such embodiments, the membrane anchored immunomodulatory agent is tethered to the TIL surface membrane by a cell membrane anchor moiety, thereby allowing the immunomodulatory agent to exert its effect in a targeted manner.

The immunomodulator may be any suitable immunomodulator, including, for example, any of the immunomodulators provided herein. In some embodiments, the immunomodulator is an interleukin that promotes an anti-tumor response. In some embodiments, the immunomodulatory agent is a cytokine. In particular embodiments, the immunomodulator is an IL-2, IL-12, IL-15, IL-18, IL-21, or CD40 agonist (e.g., CD40L or an agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)) or biologically active variant thereof. In certain embodiments, two or more different membrane-anchored immunomodulatory fusion proteins are expressed on the surface of the TIL. In exemplary embodiments, the TIL comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different membrane-anchored immunomodulatory fusion proteins.

The immunomodulator is linked to a cell membrane anchor portion that tethers the immunomodulator to the surface of the TIL cells. Suitable cell membrane anchor moieties include, for example, transmembrane domains of endogenous TIL cell surface proteins and fragments thereof. Exemplary transmembrane domains useful in the fusion proteins of the invention include, for example, B7-1, B7-2 and CD8a transmembrane domains, and fragments thereof. In some embodiments, the cell membrane anchor portion further comprises transmembrane and intracellular domains of endogenous TIL cell surface proteins or fragments thereof. In some embodiments, the cell membrane anchor moiety is a B7-1, B7-2 or CD8a transmembrane-intracellular domain or fragment thereof. In certain embodiments, the cell membrane anchor portion is a CD8a transmembrane domain having the amino acid sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 238). In certain embodiments, the cell membrane anchor portion is a B7-1 transmembrane-intracellular domain having the amino acid sequence of LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 239). In certain embodiments, the cell membrane anchor moiety is a non-peptide cell membrane anchor moiety. In an exemplary embodiment, the non-peptide cell membrane anchor moiety is a glycophospholipid inositol (GPI) anchor. The GPI anchor has a structure comprising a phosphoethanolamine linker, a glycan core, and a phospholipid tail. In some embodiments, the glycan core has more than one side chain modification. In some embodiments, the glycan core has one or more modifications in the following side chains: phosphoethanolamine groups, mannose, galactose, sialic acid, or other sugars.

Membrane anchored immunomodulatory fusion proteins include linkers that enable the attachment of components of the membrane anchored immunomodulatory fusion protein (e.g., immunomodulatory agents to cell membrane anchor portions). Suitable linkers include linkers of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length. In some embodiments, the length of the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, 50-60 amino acids. Suitable linkers include, but are not limited to: cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers or non-helical linkers. In some embodiments, the linker is a peptide linker, optionally comprising Gly and Ser. In certain embodiments, the peptide linker utilizes glycine-serine polymers, including, for example, (GS)n(SEQ ID NO：240)、(GSGGS)n(SEQ ID NO：241)、(GGGS)n(SEQ ID NO：242)、(GGGGS)n(SEQ ID NO：243)、(GGGGGS)n(SEQ ID NO：244) and (GGGGGGS) n (SEQ ID NO: 245), where n is an integer of at least one (and typically 3 to 10). Other linkers that may be used with the compositions and methods of the present invention are described in U.S. patent publication nos. US 2006/007488, US 20050238649, and US2006/0024317, each of which is incorporated herein by reference in its entirety (especially in relevant parts relating to linkers). In some embodiments, the peptide linker is SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 246).

In some embodiments, the linker is a cleavable linker. In exemplary embodiments, the cleavable linker allows release of the immunomodulatory agent into the tumor microenvironment. Cleavable linkers are also suitable for embodiments in which two membrane-anchored immunomodulatory fusion proteins are co-expressed in the same TIL (see, e.g., fig. 36 and tables 58 and 59). In an exemplary embodiment, the linker is a self-cleaving 2A peptide. See, e.g., liu et al, (sci.rep.) 7 (1): 2193 (2017), the relevant portion of which is related to the 2A peptide, is herein incorporated by reference. The 2A peptide is a viral oligopeptide that mediates cleavage of the polypeptide during translation in eukaryotic cells. In some embodiments, the 2A peptide includes a C-terminus having amino acid sequence GDVEXiNPGP (SEQ ID NO: 247), where Xi is any naturally occurring amino acid residue. In certain embodiments, the 2A peptide is porcine teschovirus-1 2A peptide (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 248). In some embodiments, the 2A peptide is a equine rhinitis A virus 2A peptide (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO: 249). In certain embodiments, the 2A peptide is a foot and mouth disease virus 2A peptide: (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 250). In some embodiments, the cleavable linker comprises a furin cleavable sequence. Exemplary furin cleavable sequences are described, for example, in Duckert et al, protein engineering, design & Selection (Protein Engineering), 17 (1): 107-112 (2004) and U.S. patent No. 8,871,906, each of which is incorporated herein by reference, particularly as regards relevant portions of the furin cleavable sequences. In some embodiments, the linker comprises a 2A peptide and a furin cleavable sequence. In an exemplary embodiment, the furin cleavable 2A peptide comprises amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 251).

In some embodiments, the immunomodulatory agent is linked in the membrane-anchored immunomodulatory fusion protein via a degradable linker (e.g., disulfide bond linker) such that the linker degrades under physiological conditions, thereby releasing the immunomodulatory agent. In some embodiments, the immunomodulatory agent is reversibly linked to the functional group through a degradable linker such that the linker degrades under physiological conditions and releases the immunomodulatory agent. Suitable degradable linkers include, but are not limited to: protease-sensitive linkers that are sensitive to one or more enzymes present in the biological medium, such as proteases in the tumor microenvironment, matrix metalloproteinases (e.g., matrix metalloproteinase 2 (MMP 2) or matrix metalloproteinase 9 (MMP 9)) present in, for example, tumor microenvironment or inflamed tissue.

In other embodiments, the components of the membrane-anchored immunomodulatory fusion protein are linked by an enzyme-sensitive linker. Exemplary cleavable linkers include those recognized by one of the following enzymes: metalloproteinases MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. See, for example, U.S. patent nos. 8,541,203 and 8,580,244, the entire contents of which and the relevant parts relating to cleavable linkers are each incorporated herein by reference.

In certain embodiments, the membrane-anchored immunomodulatory fusion protein comprises a signal peptide that facilitates translocation of the fusion protein to the TIL cell membrane. Any suitable signal peptide that facilitates localization of the fusion protein to the TIL cell membrane may be used. In some embodiments, the signal peptide does not interfere with the biological activity of the immunomodulator. Exemplary signal peptide sequences include (but are not limited to): human granulocyte-macrophage colony-stimulating factor (GM-CSF), a receptor signal sequence, a human prolactin signal sequence, and a human IgE signal sequence. In certain embodiments, the fusion protein comprises a human IgE signal sequence. In an exemplary embodiment, the human IgE signal sequence has amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 252). In some embodiments, the human IgE signal sequence includes amino acid sequence NIKGSPWKGSLLLLLVSNLLLCQSVAP (SEQ ID NO: 253). In some embodiments, the signal peptide sequence is an IL-2 signal sequence having amino acid sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 254).

In some embodiments, the membrane-anchored immunomodulatory fusion protein is according to the formula:

S-IA-L-C，

Wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety.

In some embodiments, the signal peptide S is SEQ ID NO: 252-254. In some embodiments, the cell membrane anchor portion is SEQ ID NO:277. in exemplary embodiments, the immunomodulator is an IL-2, IL-12, IL-15, IL-18, IL-21, or CD40 agonist (e.g., CD40L or anti-CD 40 scFv as described herein). In some embodiments, C is a B7-1 transmembrane-intracellular domain (e.g., SEQ ID NO: 239). Exemplary membrane-anchored immunomodulatory fusion proteins according to the above formula are depicted in fig. 36 and 37.

In some embodiments, the TIL comprises two or more different membrane-anchored immunomodulatory fusion proteins from N-terminus to C-terminus according to the formula: S-IA-L-C, wherein each of the different membrane anchored immunomodulatory fusion proteins comprises a different immunomodulatory agent. In some embodiments, the two or more different immunomodulatory agents are selected from: IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12.

In some embodiments comprising two membrane-anchored immunomodulatory fusion proteins, the membrane-anchored immunomodulatory fusion proteins are arranged from N-terminus to C-terminus according to the following formula:

S1-IA1-L1-C1-L2-S2-IA2-L3-C2，

Wherein S1 and S2 are each a signal peptide, IA1 and IA2 are each immunomodulators, L1-L3 are each a linker, and C1 and C2 are each a cell membrane anchor moiety. In some embodiments, IA1 and IA2 are the same immunomodulator. In certain embodiments, IA1 and IA2 are different immunomodulators. Suitable immunomodulators include any of the immunomodulators described herein. In some embodiments, IA1 and IA2 are independently selected from IL-2, IL-12, IL-15, IL-18, IL-21, CD40 agonists (e.g., CD40L or an agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)) or biologically active variants thereof. In some embodiments, IA1 and IA2 are selected from: IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12. In some embodiments, more than one of L1 to L3 is a cleavable linker. In some embodiments, two or more of L1 to L3 are different linkers. In an exemplary embodiment, L2 is a cleavable linker. In some embodiments, L2 is a furin cleavable P2A linker (e.g., SEQ ID NO: 251). In some embodiments, C1 and C2 are independently a transmembrane domain and/or a transmembrane-intracellular domain. In certain embodiments, C1 and C2 are the same. In an exemplary embodiment, C1 and C2 are each a B7-1 transmembrane-intracellular domain (e.g., SEQ ID NO: 239). In an exemplary embodiment, C1 and C2 are different. Exemplary constructs comprising two membrane-anchored immunomodulatory fusion proteins according to the above formula are depicted in figure 36 and tables 58 and 59.

Modified TILs comprising cell membrane anchored immunomodulatory fusion proteins bound to a surface can be prepared by genetically modifying a population of TILs to comprise nucleic acids encoding the fusion proteins. Any suitable genetic modification method can be used to produce such modified TILs, including, for example, CRISPR, TALE, and zinc finger methods described herein.

Any suitable population of TILs may be genetically modified to produce a modified TIL composition of the invention. In some embodiments, the population of TILs produced during any step of the process 2A method of the invention (see, e.g., fig. 2-6) is genetically modified to produce modified TILs of the invention. In an exemplary embodiment, the population of TILs produced during any step of the GEN 3 method of the invention (see, e.g., fig. 7) is genetically modified to produce modified TILs of the invention. In exemplary embodiments, the TIL produced by the second step in the process 2A method and/or the rapid amplification step in the GEN 3 method provided herein is genetically modified to produce a modified TIL of the invention. In some embodiments, PD-1 positive TILs that have been preselected using the methods described herein are genetically modified to produce modified TILs of the invention.

Any suitable population of TILs may be transiently modified to produce transiently modified TIL compositions of the invention. In some embodiments, the population of TILs produced during any step of the process 2A methods of the invention (see, e.g., fig. 2-6) encodes a nucleic acid transfection of a cell membrane anchored immunomodulatory fusion protein to transiently express the cell membrane anchored immunomodulatory fusion protein in the transiently modified TILs of the invention. In an exemplary embodiment, the population of TILs produced during any step of the GEN 3 method of the invention (see, e.g., fig. 7) encodes a nucleic acid transfection of a cell membrane anchored immunomodulatory fusion protein to transiently express the cell membrane anchored immunomodulatory fusion protein in the transiently modified TILs of the invention. In an exemplary embodiment, the TIL produced by the first amplification step in the process 2A method and/or the initial amplification step in the GEN 3 method provided herein encodes a nucleic acid transfection of a cell membrane anchored immunomodulatory fusion protein to transiently express the cell membrane anchored immunomodulatory fusion protein in the transiently modified TIL of the invention. In an exemplary embodiment, the TIL produced by the second amplification step in the process 2A method and/or the rapid amplification step in the GEN 3 method provided herein encodes a nucleic acid transfection of a cell membrane anchored immunomodulatory fusion protein to transiently express the cell membrane anchored immunomodulatory fusion protein in the transiently modified TIL of the invention. In some embodiments, the PD-1 positive TIL that has been preselected using the methods described herein encodes a nucleic acid transfection of a cell membrane anchored immunomodulatory fusion protein to transiently express the cell membrane anchored immunomodulatory fusion protein in a transiently modified TIL of the invention.

Also provided herein are nucleic acids encoding membrane-anchored immunomodulatory fusion proteins, expression vectors comprising such nucleic acids, and host cells comprising the nucleic acids or expression vectors. Any suitable promoter may be used for the expression of the membrane anchored immunomodulatory fusion protein. In exemplary embodiments, the promoter is an inducible promoter. Exemplary nucleic acids encoding exemplary membrane-anchored immunomodulatory fusion proteins and components of such fusion proteins are depicted in fig. 36 and 37, as well as tables 58 and 59.

In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is mRNA. In exemplary embodiments, the mRNA has one or more modifications that improve the intracellular stability and/or translational efficiency of the mRNA. In some embodiments, the mRNA has a 5' cap (cap) or cap analogue that improves the half-life of the mRNA. Exemplary cap structures include, but are not limited to, ARCA, mCAP, m ⁷ gppppn (cap 0), m ⁷ GpppNm (cap 1), and m ⁷ GPPPNMPNM (cap 2) caps. In some embodiments, the 5' cap is according to the formula: ^m7Gppp[N₂'_Ome]_n[N]_m Wherein ^m7 G is N7-methylated guanosine or any guanosine analogue, N is any natural, modified or unnatural nucleoside, N can be any integer from 0 to 4, and m can be an integer from 1 to 9. Exemplary 5 'caps are disclosed in U.S. patent No. 10,703,789 and WO2017053297, which are incorporated herein by reference in their entirety, particularly in connection with 5' caps and cap analogues.

In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is an mRNA, which further comprises a 3' untranslated region (UTR) or a modified UTR. 3' UTRs are known to have extensions of adenosine and uridine. These AU-rich markers are particularly prevalent in genes with high conversion rates. Based on their sequence characteristics and functional properties, AU-rich elements (ARE) can be divided into three classes (Chen et al, 1995): class I ARE contain several copies of the discrete AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have more than two overlapping UUAUUUA (U/A) (U/A) nonamers. Molecules containing ARE of this type include GM-CSF and TNF-a. Class III ARE less well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin (myogenin) are two well studied examples of this class. Most of the proteins that bind ARE known to destabilize the messenger, whereas ELAV family members (most notably HuR) have been reported to increase mRNA stability. HuR binds to ARE of all three classes. Engineering a HuR specific binding site into the 3' utr of a nucleic acid molecule will result in HuR binding, thus leading to stabilization of in vivo information.

The introduction, removal or modification of 3' utr AU-rich elements (ARE) can be used to modulate the stability of nucleic acids described herein. When engineering a particular nucleic acid, more than one copy of an ARE can be introduced to make the polynucleotides of the invention more labile, thereby reducing translation and reducing production of the resulting protein. Similarly, ARE can be identified and removed or mutated to increase intracellular stability, thereby increasing translation and production of the resulting protein. Transfection experiments can be performed using nucleic acids in relevant cell lines, and protein production can be analyzed at various time points after transfection. For example, cells can be transfected with different ARE engineering molecules and the resulting proteins analyzed 6 hours, 12 hours, 24 hours, 48 hours and 7 days post-transfection by using an ELISA kit for the relevant proteins.

In some embodiments, the nucleic acid encoding the membrane anchored immunomodulatory fusion protein is operably linked to an activated T cell Nuclear Factor (NFAT) promoter, or a functional portion or functional variant thereof. As used herein, "NFAT promoter" refers to more than one NFAT responsive element linked to the minimal promoter of any gene expressed by a T cell. Preferably, the minimal promoter of the gene expressed by the T cell is the minimal human IL-2 promoter. The NFAT response element may comprise, for example, nfatt, NFAT2, NFAT3 and/or NFAT4 response elements. The NFAT promoter (or functional portion or functional variant thereof) may comprise any number of binding motifs, for example at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at most twelve binding motifs.

Table 4: NFAT promoter related sequences

In a preferred embodiment, the NFAT promoter comprises six NFAT binding motifs. See, for example, U.S. patent No. 8,556,882, which is incorporated herein by reference in its entirety (especially in the relevant part relating to the NFAT promoter). In some embodiments, the NFAT promoter system controls expression of an immunomodulatory fusion protein comprising any of the immunomodulatory agents described herein. In certain embodiments, the immunomodulator is selected from the group consisting of: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)) or biologically active variants thereof. Exemplary nucleic acids encoding exemplary membrane-anchored immunomodulatory fusion proteins of the invention operably linked to NFAT promoters are depicted in table 59. In some embodiments, the NFAT promoter system controls expression of an immunomodulatory fusion protein comprising IL-15. In some embodiments, the NFAT promoter system controls expression of immunomodulatory fusion proteins comprising IL-21. In some embodiments, the NFAT promoter system controls the expression of immunomodulatory fusion proteins comprising IL-15 and IL-21.

In some embodiments, the invention provides a TIL that is genetically modified to comprise DNA encoding an immunomodulatory fusion protein operably linked to an NFAT promoter. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising any of the immunomodulatory agents described herein. In certain embodiments, the immunomodulator is selected from the group consisting of: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)) or biologically active variants thereof. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising IL-15. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising IL-21. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising IL-15 and IL-21.

In some embodiments, the invention provides a TIL genetically modified to comprise DNA encoding an immunomodulatory fusion protein operably linked to an NFAT promoter, wherein the immunomodulatory fusion protein is arranged from N-terminus to C-terminus according to the formula:

S1-IA1-L1-C1-L2-S2-IA2-L3-C2，

Wherein S1 and S2 are each a signal peptide, IA1 and IA2 are each immunomodulators, L1 to L3 are each a linker, and C1 and C2 are each a cell membrane anchor moiety. In some embodiments, IA1 and IA2 are the same immunomodulator. In certain embodiments, IA1 and IA2 are different immunomodulators. Suitable immunomodulators include any of the immunomodulators described herein. In some embodiments, IA1 and IA2 are independently selected from IL-2, IL-12, IL-15, IL-18, IL-21, CD40 agonists (e.g., CD40L or agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)) or biologically active variants thereof. In some embodiments, IA1 and IA2 are selected from: IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12. In some embodiments, IA1 and IA2 are independently selected from IL-15 and IL-21. In some embodiments, IA1 is IL-15 and IA2 is IL-21. In some embodiments, IA1 is IL-21 and IA2 is IL-15. In some embodiments, more than one of L1 to L3 is a cleavable linker. In some embodiments, two or more of L1 to L3 are different linkers. In an exemplary embodiment, L2 is a cleavable linker. In some embodiments, L2 is a furin cleavable P2A linker (e.g., SEQ ID NO: 251). In some embodiments, C1 and C2 are independently a transmembrane domain and/or a transmembrane-intracellular domain. In certain embodiments, C1 and C2 are the same. In an exemplary embodiment, C1 and C2 are each a B7-1 transmembrane-intracellular domain (e.g., SEQ ID NO: 239). In an exemplary embodiment, C1 and C2 are different. An exemplary construct comprising two membrane-anchored immunomodulatory fusion proteins according to the above formula is depicted in fig. 36.

The nucleic acid encoding the membrane-anchored immunomodulatory fusion proteins of the invention may be introduced into a population of TILs using any suitable method to produce transiently modified or genetically modified TILs that express the membrane-anchored immunomodulatory fusion proteins. In some embodiments, a nucleic acid encoding a membrane anchored immunomodulatory fusion protein is introduced into the TIL population using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. See, for example, international patent application publication No. WO 2013/059343A1, WO 2017/008063A1, or WO 2017/123663A1, or U.S. patent application publication No. US2014/0287509A1, US2018/0201889A1, or US2018/0245089A1, each of which is incorporated herein by reference in its entirety (especially with respect to the disclosure of microfluidic platforms for nucleic acid delivery). In the SQZ platform, delivery of nucleic acids encoding membrane-anchored immunomodulatory fusion proteins into cells is achieved by the transient disruption of the cell membrane of cells for modification (e.g., TIL) by microfluidic contraction.

In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is mRNA, and the mRNA is delivered into the TIL using a microfluidic platform (e.g., a SQZ vector-free microfluidic platform) to produce a transiently modified TIL. In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is DNA, and the DNA is delivered into the TIL using a microfluidic platform (e.g., a SQZ vector-free microfluidic platform) to produce a stable genetically modified TIL. Nucleic acids may be delivered using a microfluidic platform (e.g., a SQZ carrier-free microfluidic platform) into any population of TILs generated during any step of the process 2A method of the invention (see, e.g., fig. 2-6) or the GEN 3 method of the invention (see, e.g., fig. 7) to generate modified TILs. In some embodiments, the membrane anchored immunomodulatory fusion protein comprises IL-2, IL-12, IL-15, IL-18, IL-21, a CD40 agonist (e.g., CD40L or an agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)), or any combination thereof.

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-15. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-18, IL-21, CD40L, or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is CD40L. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, IL-21, a CD40 agonist (e.g., CD40L or an agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)) or a biologically active variant thereof.

In exemplary embodiments, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12. In some embodiments, the second immunomodulatory agent is IL-2, IL-15, IL-18, IL-21, CD40L, or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-18. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-21, CD40L, or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-21. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, CD40L, or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, IL-21, CD40L, or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-12.

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-15.

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-18.

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-21.

In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is CD40L or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is IL-15.

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is IL-18.

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is IL-21.

In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is CD40L or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-15 and the second immunomodulatory agent is IL-18.

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-15 and the second immunomodulatory agent is IL-21.

In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-15 and the second immunomodulatory agent is CD40L or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In an exemplary embodiment, the modified TIL provided herein comprises two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-18 and the second immunomodulatory agent is IL-21.

In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-18 and the second immunomodulatory agent is CD40L or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-21 and the second immunomodulatory agent is CD40L or an anti-CD 40 binding domain (e.g., an anti-CD 40 scFv).

Other membrane-anchored immunomodulatory fusion proteins that may be included in the modified TILs provided herein are described in WO 2019/157130A1, which is incorporated herein by reference in its entirety (particularly in the relevant part relating to membrane-anchored immunomodulatory fusion proteins).

Exemplary membrane-anchored immunomodulatory fusion proteins comprised in the modified TILs provided herein are depicted in fig. 36 and 37, and tables 58 and 59.

In some embodiments, a nucleic acid encoding any of the membrane-anchored immunomodulatory fusion proteins described above is operably linked to an NFAT promoter, or a functional portion or functional variant thereof.

2. Immunomodulatory agent-TIL antigen binding domain fusion proteins

In some embodiments, the modified TILs provided herein comprise immunomodulatory fusion proteins, wherein such fusion proteins comprise one or more immunomodulatory agents linked to a TIL Antigen Binding Domain (ABD). In some embodiments, after the TIL ABD binds to the TIL surface antigen, the one or more immunomodulatory agents are tethered to the TIL surface membrane.

The TIL antigen binding domain comprises an antibody heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the TIL antigen binding domain is a full length antibody comprising a heavy chain according to the formula VH-CH 1-hinge-CH 2-CH3 and a light chain according to the formula VL-CL, wherein VH is a heavy chain variable domain, CH1, CH2, CH3 are heavy chain constant domains, VL is a light chain variable domain, and CL is a light chain constant domain. In some embodiments, the TIL antigen binding domain is an antibody fragment. In certain embodiments, the TIL antigen binding domain is Fab, fab ', F (ab') 2, F (ab) 2, a variable fragment (Fv), a domain antibody (dAb), or a single chain variable fragment (scFv).

The TIL antigen binding domain may bind any suitable TIL antigen that can effect the attachment of the immunomodulatory agent-TIL ABD fusion protein to the surface of the TIL. In exemplary embodiments, the TIL antigen binding domain is capable of binding to a TIL surface antigen. TIL surface antigens include (but are not limited to )D16、CD45、CD4、CD8、CD3、CD11a、CD11b、CD11c、CD18、LFA-1、CD25、CD127、CD56、CD19、CD20、CD22、HLA-DR、CD197、CD38、CD27、CD137、OX40、GITR、CD56、CD196、CXCR3、CXCR4、CXCR5、CD84、CD229、CCR1、CCR5、CCR4、CCR6、CCR8 and/or ccr10. In some embodiments, ABD binds CD45. In particular embodiments, ABD binds CD45 isoforms selected from CD45RA, CD45RB, CD45RC, or CD45rβ.

In certain embodiments, ABD binds a checkpoint inhibitor. Exemplary checkpoint inhibitors include, but are not limited to, PD-1, PD-L1, LAG-3, TIM-3 and CTLA-4 (see, e.g., qin et al, molecular Cancer 18:155 (2019)). In some embodiments, the ABD binds to a checkpoint inhibitor expressed on immune effector cells (e.g., T cells or NK cells). Exemplary anti-PD-1 antibodies are disclosed, for example, in U.S. patent nos. US 7,695,715, US 7,332,582, US 9,205,148, US 8,686,119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731, and US 9,102,727, which are incorporated herein by reference in their entirety (especially in the relevant parts relating to anti-PD-1 antibodies). Exemplary anti-PD-L1 antibodies are disclosed in U.S. patent nos. US 8,217,149, US 8,779,108, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082, which are incorporated herein by reference in their entirety (especially in the relevant parts relating to anti-PD-L1 antibodies). Exemplary anti-LAG-3 antibodies are disclosed in U.S. patent nos. US 9,244,059, US 9,244,059, and US 9,505,839, which are incorporated herein by reference in their entirety (especially in the relevant part relating to anti-LAG-3 antibodies). Exemplary TIM-3 antibodies are disclosed in WO 2016/161270, US 8,841,418, and US 9,163,087, which are incorporated herein by reference in their entirety, particularly in the relevant part in relation to anti-TIM-3 antibodies. Exemplary CTLA-4 antibodies are disclosed in US 6,984,720 and US 7,411,057, which are incorporated herein by reference in their entirety, particularly in the relevant sections relating to anti-CTLA-4 antibodies.

In some embodiments, the ABD is an anti-CD 45 antibody or fragment thereof. In certain embodiments, the anti-CD 45 antibody is a human anti-CD 45 antibody, a humanized anti-CD 45 antibody, or a chimeric anti-CD 45 antibody. In an exemplary embodiment, the ABD comprises vhCDR-3 and vlCDR-1-3 of the anti-CD 45 antibody BC8 (see US20170326259, which is incorporated herein by reference, particularly relevant parts related to the anti-CD 45 antibody sequence). In some embodiments, the ABD comprises a heavy chain variable domain and a variable domain of an anti-CD 45 antibody BC 8. In some embodiments, the ABD comprises VhCDR1-3 and vlCDR1-3 or VH and VL of one of the following anti-CD 45 antibodies: 10G10, UCHL1, 9.4, 4B2 or GAP8.3 (see Spertini et al, immunology 113 (4): 441-452 (2004), buzzi et al, cancer research CANCER RESEARCH, 52:4027-4035 (1992)).

The immunomodulatory fusion protein may be any suitable immunomodulatory agent, including, for example, any immunomodulatory agent provided herein. In some embodiments, the immunomodulator is an interleukin that promotes an anti-tumor response. In some embodiments, the immunomodulatory agent is a cytokine. In particular embodiments, the immunomodulator is IL-2, IL-12, IL-15, IL-21, or a biologically active variant thereof. In certain embodiments, the fusion protein comprises more than one immunomodulatory agent. In exemplary embodiments, the fusion protein comprises 2,3,4, 5, 6, 7, 8, 9, or 10 different immunomodulatory agents.

The TIL antigen binding domain is attached to the immunomodulatory agent using any suitable linker. Suitable linkers include (but are not limited to): cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers or non-helical linkers. In some embodiments, the linker is a peptide linker, optionally comprising Gly and Ser. Suitable linkers include linkers of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, or 50-60 amino acids in length. In certain embodiments, the peptide linker is a (GGGS) _n or (GGGGS) _n linker, wherein n indicates the number of repeats of the motif and is an integer selected from 1 to 10. In some embodiments, the linker is an antibody hinge domain or fragment thereof. In certain embodiments, the linker is a human immunoglobulin (Ig) hinge domain (e.g., an IgG1, igG2, igG3, igG4, igD, igE, igM, or IgA hinge) or fragment thereof. In some embodiments, the immunomodulatory agent is directly coupled to the TIL without the presence of a linker.

The immunomodulator may be attached to the TIL antigen binding domain at a suitable position that does not interfere with binding of the fusion protein to the TIL. In some embodiments in which the antigen binding domain is a full length antibody, the immunomodulator is attached to the C-terminus or N-terminus of the heavy or light chain. In some embodiments in which the antigen binding domain is an scFv, the immunomodulatory agent is linked to the C-terminus or N-terminus of the heavy chain variable domain or the light chain variable domain. In some embodiments in which the antigen binding domain is a Fab, the immunomodulatory agent is linked to the C-terminus or N-terminus of the heavy chain variable domain or the light chain variable domain. In some embodiments in which the antigen binding domain is a Fab', the immunomodulator is linked to the C-terminus or N-terminus of the heavy chain variable domain or the light chain variable domain. In some embodiments where the antigen binding domain is Fab' ₂, the immunomodulatory agent is linked to the C-terminus or N-terminus of the heavy chain variable domain or the light chain variable domain.

In some embodiments where the fusion protein comprises more than two immunomodulators, any of the immunomodulators described herein are used to interconnect the immunomodulators. In some embodiments, two or more immunomodulators are attached to different positions of the antigen binding domain. For example, in some embodiments in which the TIL antigen binding domain is a full length antibody, two or more immunomodulators are linked at the following positions: (i) different positions on the heavy chain, (ii) different positions on the light chain, or (iii) different positions on the heavy chain and/or the light chain.

The immunomodulatory agent-TIL antigen binding domain fusion proteins of the invention may be prepared using any suitable method. In one aspect, provided herein are nucleic acids encoding the fusion proteins of the invention, expression vectors comprising such nucleic acids, and host cells comprising the expression vectors. Host cells comprising an expression vector encoding the fusion protein of the invention are cultured under conditions for expression of the fusion protein, and the fusion protein is then isolated and purified. In some embodiments, the purified fusion protein is then incubated with a population of TILs under conditions that effect binding of the fusion protein to TILs.

In some embodiments, an immunomodulatory agent-TIL antigen binding domain fusion protein of the invention is linked to a TIL produced during any step of the process 2A method of the invention (see, e.g., figures 2-6). In an exemplary embodiment, the fusion protein is linked to the TIL produced during any step of the GEN 3 method of the invention (see, e.g., fig. 7). In exemplary embodiments, the fusion protein is linked to the TIL produced by the first amplification step in the process 2A method and/or the initial amplification step in the GEN 3 method provided herein. In exemplary embodiments, the fusion protein is linked to the TIL produced by the second amplification step in the process 2A method and/or the rapid amplification step in the GEN 3 method provided herein. In some embodiments, the TIL is a PD-1 positive TIL that has been pre-selected using the methods described herein.

Nucleic acids encoding the immunomodulatory agent-TIL antigen-binding domain fusion proteins of the invention can be introduced into a population of TILs using any suitable method to produce transiently modified or genetically modified TILs that express the immunomodulatory agent-TIL antigen-binding domain fusion proteins of the invention. In some embodiments, a nucleic acid encoding an immunomodulatory agent-TIL antigen binding domain fusion protein of the invention is introduced into a population of TILs using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. See, for example, international patent application publication No. WO 2013/059343A1, WO 2017/008063A1, or WO 2017/123663A1, or U.S. patent application publication No. US2014/0287509A1, US2018/0201889A1, or US2018/0245089A1, each of which is incorporated herein by reference in its entirety, especially with respect to the disclosure of microfluidic platforms for nucleic acid delivery. In the SQZ platform, delivery of nucleic acids encoding immunomodulatory agent-TIL antigen binding domain fusion proteins into cells is achieved by the microfluidic contraction to temporarily disrupt the cell membrane of the cell (e.g., TIL) for modification.

In some embodiments, the nucleic acid encoding an immunomodulatory agent-TIL antigen binding domain fusion protein of the invention is mRNA, and the mRNA is delivered into the TIL using a microfluidic platform (e.g., a SQZ carrier-free microfluidic platform) to produce a transiently modified TIL. In some embodiments, the nucleic acid encoding the immunomodulatory agent-TIL antigen binding domain fusion proteins of the invention is DNA, and the nucleic acid is delivered into the TIL using a microfluidic platform (e.g., an SQZ vector-free microfluidic platform) to produce a stable genetically modified TIL. Nucleic acids may be delivered using a microfluidic platform (e.g., an SQZ carrier-free microfluidic platform) into any population of TILs generated during any step of the process 2A method of the invention (see, e.g., fig. 2-6) or the GEN 3 method of the invention (see, e.g., fig. 7) to generate modified TILs. In some embodiments, the membrane anchored immunomodulatory fusion protein comprises IL-2, IL-12, IL-15, IL-21, or a combination thereof (e.g., IL-15 and IL-21).

Exemplary immunomodulatory-TIL antigen-binding domain fusion proteins suitable for use in the compositions and methods provided herein are also described, for example, in U.S. patent application publication No. 20200330514, the entire contents of which and the relevant portions relating to immunomodulatory-TIL antigen-binding domain fusion proteins are incorporated herein by reference.

B. Nanoparticle compositions

In some embodiments, the modified TILs of the present invention provided herein comprise more than one nanoparticle comprising more than one immunomodulatory agent. In some embodiments, the nanoparticles provided herein comprise a plurality of two or more proteins coupled to each other and/or to a second component of the particle (e.g., reversibly linked by a degradable linker). In some embodiments, the nanoparticle protein is present in a polymer or silica. In certain embodiments, the nanoparticle comprises a nanoshell. The nanoparticles provided herein comprise more than one immunomodulator. In some embodiments, the immunomodulator is IL-2, IL-12, IL-15, IL-18, IL-21, a CD40 agonist (e.g., CD40L or an agonistic anti-CD 40 binding domain (e.g., anti-CD 40 scFv)) or a biologically active variant thereof. The nanoparticles are attached to the surface of the TIL using any suitable technique described herein.

Exemplary nanoparticles for use in the modified TILs of the present invention provided herein include, but are not limited to, liposomes, protein nanogels, nucleotide nanogels, polymer nanoparticles, or solid state nanoparticles. In some embodiments, the nanoparticle comprises a liposome. In an exemplary embodiment, the nanoparticle comprises an immunomodulatory agent nanogel. In certain embodiments, the nanoparticle is an immunomodulatory agent nanogel having a plurality of immunomodulatory agents (e.g., cytokines) covalently linked to one another. In some embodiments, the nanoparticle comprises at least one polymer, cationic polymer, or cationic block copolymer on the surface of the nanoparticle. Exemplary nanoparticles useful in the compositions provided herein are disclosed, for example, in U.S. patent nos. 9,283,184 and 9,603,944, the entire contents of which and the relevant portions relating to the nanoparticles are each incorporated herein by reference.

The immunomodulator may be any suitable immunomodulator, including, for example, any of the immunomodulators provided herein. In some embodiments, the immunomodulator is an interleukin that promotes an anti-tumor response. In some embodiments, the immunomodulatory agent is a cytokine. In particular embodiments, the immunomodulator is IL-2, IL-12, IL-15, IL-21, or a biologically active variant thereof. In certain embodiments, the fusion protein comprises more than one immunomodulatory agent. In exemplary embodiments, the fusion protein comprises 2,3, 4, 5, 6, 7, 8, 9, or 10 different immunomodulatory agents.

In some embodiments, the nanoparticle comprises proteins that are covalently cross-linked to each other and/or to a second component (e.g., a degradable linker). In some embodiments, the nanoparticle comprises an immunomodulatory agent linked to a functional group or polymer or "reversibly modified" through a degradable linker. In some embodiments, the nanoparticle is a nanogel comprising a plurality of immunomodulators that are cross-linked to each other by a degradable linker (see U.S. patent No. 9,603,944). In an exemplary embodiment, the protein of the nanogel is crosslinked with a polymer (e.g., polyethylene glycol (PEG)). In some embodiments, the polymer is surface crosslinked with the nanogel.

In some embodiments, the immunomodulatory agents of the nanoparticles are reversibly linked to each other by a degradable linker (e.g., disulfide bond linker) such that the linker degrades under physiological conditions, thereby releasing the immunomodulatory agents. In some embodiments, the immunomodulatory agent of the nanoparticle is reversibly linked to the functional group through a degradable linker such that the linker degrades under physiological conditions and releases the immunomodulatory agent. Suitable degradable linkers include (but are not limited to): two N-hydroxysuccinimide (NHS) ester groups joined together by a flexible disulfide-containing linker that is sensitive to the reducing physiological environment; a hydrolyzable linker that is sensitive to an acidic physiological environment (pH <7, e.g., pH of 4 to 5, 5 to 6, or 6 to less than 7, e.g., pH of 6.9), or a protease sensitive linker that is sensitive to more than one enzyme present in a biological medium (e.g., a protease in a tumor microenvironment, such as a matrix metalloproteinase present in a tumor microenvironment or inflamed tissue (e.g., matrix metalloproteinase 2 (MMP 2) or matrix metalloproteinase 9 (MMP 9))). A crosslinking agent sensitive to a reducing physiological environment is, for example, a crosslinking agent having a disulfide bond containing linker that will react with amino groups on the protein by the presence of NHS groups, thereby crosslinking the protein into a high density protein nanogel. In some embodiments, the degradable crosslinking agent comprises bis [2- (N-succinimidyl-oxycarbonyloxy) ethyl ] disulfide.

In some embodiments, the degradable linker comprises at least one N-hydroxysuccinimide ester. In some embodiments, the degradable linker is a redox-reactive linker. In some embodiments, the redox-reactive linker comprises a disulfide bond. In some embodiments, the degradable linkers provided herein comprise at least one N-hydroxysuccinimide ester capable of reacting with a protein at neutral pH (e.g., about 6 to about 8, or about 7) without substantially denaturing the protein. In some embodiments, the degradable linker is a "redox-reactive" linker, meaning that it degrades under physiological conditions (e.g., 20 to 40 ℃ and/or pH 4 to 8) in the presence of a reducing agent (e.g., glutathione, GSH), thereby releasing the intact protein to which it is reversibly attached from the compound. In some embodiments, the protein of the nanoparticle is attached to the degradable linker through a terminal or internal-NH ₂ functional group (e.g., a side chain of lysine).

In other embodiments, the proteins of the nanoparticles are linked by an enzyme-sensitive linker. Exemplary cleavable linkers include those recognized by one of the following enzymes: metalloproteinases MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. See, for example, U.S. patent nos. 8,541,203 and 8,580,244, the entire contents of which and the relevant parts relating to cleavable linkers are each incorporated herein by reference.

In some embodiments, the nanoparticle is a nanogel that includes a plurality of immunomodulatory agents (e.g., cytokines) that are monodisperse. In some embodiments, the immunomodulatory agent of the nanogel is crosslinked with a polymer. In certain embodiments, the polymer is crosslinked with the surface of the nanogel. In a particular embodiment, the nanogel comprises: a) More than one immunomodulator reversibly covalently cross-linked to each other via a degradable linker; and b) a polymer crosslinked with the protein exposed at the surface of the nanogel. Such nanogels may be prepared by contacting one or more immunomodulators with a degradable linker under the following conditions: the immunomodulators are allowed to covalently cross-link reversibly to each other through degradable linkers to form a variety of immunomodulator nanogels. The immunomodulator nanogel is then contacted with a polymer (e.g., polyethylene glycol) under conditions that allow the polymer to crosslink with the immunomodulator of the immunomodulator nanogel, thereby producing a plurality of immunomodulator-polymer nanogels.

In some embodiments, the nanoparticle comprises more than one polymer. Exemplary polymers include (but are not limited to): aliphatic polyesters, poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone (PCL), polyanhydrides, poly (o) esters (poly (ortho) esters), polyurethanes, poly (butyric acid), poly (valeric acid) and poly (lactide-co-caprolactone), and natural polymers (e.g., alginate and other polysaccharides (including polydextrose and cellulose), collagen, their chemical derivatives (including substitution, addition of chemical groups (e.g., alkyl, alkylene), hydroxylation, oxidation, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In some embodiments, the nanoparticle's immunomodulator is attached to a hydrophilic polymer. Exemplary hydrophilic polymers include (but are not limited to): polyethylene glycol (PEG), polyethylene glycol-b-polylysine (PEG-PLL) and/or polyethylene glycol-b-polyarginine (PEG-PArg).

In some embodiments, the nanoparticle (e.g., nanogel) comprises more than one polycation on its surface. Exemplary polycations for the nanoparticles of the present invention include, but are not limited to, polylysine (poly-L-lysine and/or poly-D-lysine), poly (arginine glyceryl succinate) (PAGS, arginine-based polymers), polyethylenimine, polyhistidine, polyarginine, protamine sulfate, polyethylene glycol-b-polylysine (PEG-PLL), and polyethylene glycol-g-polylysine.

In some embodiments, the nanoparticle associates with the TIL surface by electrostatic attraction to the TIL. In certain embodiments, the nanoparticle comprises a ligand having affinity for a surface molecule of the TIL (e.g., a surface protein, carbohydrate, and/or lipid).

In certain embodiments, the nanoparticle comprises an antigen binding domain that binds to a TIL surface antigen as described herein. In some embodiments, the antigen binding domain is an antibody or fragment thereof. In exemplary embodiments, the TIL surface antigen is CD45, LFA-1, CD11 a (integrin. Alpha. -L), CD 18 (integrin. Beta. -2), CD11b, CD11c, CD25, CD8 or CD4. In exemplary embodiments, the Antigen Binding Domain (ABD) is an anti-CD 45 antibody or fragment thereof. In certain embodiments, the anti-CD 45 antibody is a human anti-CD 45 antibody, a humanized anti-CD 45 antibody, or a chimeric anti-CD 45 antibody. In an exemplary embodiment, the ABD comprises vhCDR-3 and vlCDR-1-3 of the anti-CD 45 antibody BC8 (see US20170326259, which is incorporated herein by reference, particularly relevant parts related to the anti-CD 45 antibody sequence). In some embodiments, the ABD comprises a heavy chain variable domain and a variable domain of an anti-CD 45 antibody BC 8. In some embodiments, the ABD comprises VhCDR1-3 and vlCDR1-3 or VH and VL of one of the following anti-CD 45 antibodies: 10G10, UCHL1, 9.4, 4B2 or GAP8.3 (see Spertini et al, immunology 113 (4): 441-452 (2004), buzzi et al, cancer research CANCER RESEARCH, 52:4027-4035 (1992)). In such embodiments, the nanoparticles are attached to the surface of the population of TILs by incubating the TILs in the presence of the nanoparticles under conditions where the nanoparticles bind to the surface of the TILs.

In some embodiments, the nanoparticle is bound to the TIL cell surface by electrostatic attraction. In some embodiments, the nanoparticle is covalently bound to the TIL. In other embodiments, the nanoparticle is not covalently bound to the TIL.

In some embodiments, the nanoparticles of the present invention are attached to the TIL produced during any step of the process 2A method of the present invention (see, e.g., fig. 2-6). In an exemplary embodiment, the nanoparticle of the present invention is linked to the TIL generated during any step of the GEN 3 method of the present invention (see, e.g., fig. 7). In an exemplary embodiment, the nanoparticle of the invention is linked to the TIL produced by the first amplification step in the process 2A method and/or the initial amplification step in the GEN 3 method provided herein. In exemplary embodiments, the nanoparticle of the invention is linked to the TIL produced by the second amplification step in the process 2A method and/or the rapid amplification step in the GEN 3 method provided herein. In some embodiments, the TIL is a PD-1 positive TIL that has been pre-selected using the methods described herein.

Other suitable nanoparticles for use in the modified TILs provided herein are disclosed in U.S. patent application publication nos. US20200131239 and WO2020205808, the entire contents of which and the relevant parts relating to the nanoparticles are each incorporated herein by reference.

C. Immunomodulators

The modified TILs provided herein comprise more than one immunomodulatory agent attached to their surface. Immunomodulatory agents may be incorporated into any of the immunomodulatory fusion proteins described herein, including, for example, membrane-anchored immunomodulatory fusion proteins described herein. Any suitable immunomodulator may be included in the modified TIL of the present invention. In some embodiments, the immunomodulator enhances TIL survival and/or anti-tumor activity after transfer into the patient. Exemplary immunomodulators include, for example, cytokines. In some embodiments, the modified TIL comprises one or more ：IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、IL-23、IL-27、IL-4、IL-1α、IL-1β、IL-5、IFNγ、TNFα(TNFa)、IFNα、IFNβ、GM-CSF of the following cytokines or GCSF or biologically active variants thereof. In some embodiments, the immunomodulator is a costimulatory molecule. In a particular embodiment, the co-stimulatory molecule is one of the following: agonists of OX40, CD28, GITR, VISTA, CD, CD3, or CD 137. In some embodiments, the immunomodulator is a CD40 agonist (e.g., CD40L or an agonistic CD40 binding domain). Exemplary immunomodulators are discussed in further detail below.

1.IL-15

In some embodiments, the modified TIL provided herein comprises IL-15. In exemplary embodiments, IL-15 is included as part of an immunomodulatory fusion protein as described herein (e.g., a membrane anchored immunomodulatory fusion protein).

As used herein, "interleukin 15", "IL-15" and "IL15" all refer to interleukins that bind to and signal through a complex consisting of the IL-15 specific receptor alpha chain (IL-15 Rα), the IL-2/IL-15 receptor beta chain (CD 122) and the common gamma chain (γ -C, CD 132) (e.g., genbank accession numbers: NM-00000585, NP-000576 and NP-751915 (human), and NM-001254747 and NP-001241676 (mouse)). IL-15 has been shown to stimulate T cell proliferation within tumors. IL-15 is also capable of increasing the viability of effector memory CD8+ T cells and is important for NK cell development. Thus, without being bound by any particular theory of operation, it is believed that modified TILs associated with IL-15 described herein exhibit enhanced survival and/or anti-tumor effects.

IL-15 has a short half-life in vivo of less than 40 minutes. Modification of IL-15 monomers may improve their in vivo pharmacokinetics in the treatment of cancer. These modifications are typically focused on improving the trans-presentation of IL-15 by the alpha subunit of the IL-15 receptor (i.e., IL-15Rα). Such modifications include: 1) IL-15 is pre-associated with its soluble receptor a-subunit-Fc fusion to form an IL-15:IL-15Rα -Fc complex (see, e.g., rubenstein et al, proc. Natl. Acad. Sci. USA 103:9166-71 (2006)); 2) Expression of the superagonist IL-15-sIL-15Rα -sushi protein (see, e.g., bessard et al, molecular cancer therapeutic (Molecular cancer therapeutics) 8:2736-45 (2009)); and 3) pre-association of the human IL-15 mutant IL-15N72D with the IL-15Rα -Fc sushi-Fc fusion complex (see, e.g., zhu et al, J.Immunol. 183:3598-6007 (2009)).

In some embodiments, the IL-15 associated with the modified TIL is full-length IL-15, a fragment or variant of IL-15. In some embodiments, IL-15 is human IL-15 or a variant of human IL-15. In exemplary embodiments, IL-15 is a biologically active human IL-15 variant. In some embodiments, IL-15 and wild type IL-15 compared to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In certain embodiments, IL-15 comprises an N72D mutation as compared to wild-type human IL-15. In some embodiments, variant IL-15 exhibits IL-15Rα binding activity.

In some embodiments, the immunomodulator comprises the extracellular domains of IL-15 and IL-15Rα. In certain embodiments, the immunomodulator comprises IL-15 and IL-15Rα fused to an Fc domain (IL-15 Rα -Fc).

Table 5: related sequences of IL-15.

In some embodiments, the immunostimulatory protein is the super agonist IL-15 (IL-15 SA), which includes a complex of human IL-15 and soluble human IL-15Rα. The combination of human IL-15 with soluble human IL-15Rα forms an IL-15SA complex that has higher biological activity than human IL-15 alone. Truncated versions of the soluble human IL-15Rα and extracellular domains have been described in the art (Wei et al, 2001 J.Immunol.167:277-282). The amino acid sequence of the human IL-15Rα is shown in SEQ ID NO: 266. In some embodiments, IL-15SA includes a complex of human IL-15 with a soluble human. IL-15Rα comprises all or a portion of the extracellular domain and does not have a transmembrane or cytoplasmic domain. In some embodiments, IL-15SA includes a complex of human IL-15 with a soluble human IL-15Rα, which soluble human IL-15Rα includes a complete extracellular domain or a truncated form of an extracellular domain that retains IL-15 binding activity.

In some embodiments, IL-15SA includes a complex of human IL-15 with a soluble human IL-15Rα, which includes a truncated form of the extracellular domain that retains IL-15 binding activity. In some embodiments, the soluble human IL-15rα comprises amino acids 1 to 60, 1 to 61, 1 to 62, 1 to 63, 1 to 64, or 1 to 65 of human IL-15rα. In some embodiments, the soluble human IL-15rα comprises amino acids 1 to 80, 1 to 81, 1 to 82, 1 to 83, 1 to 84, or 1 to 85 of human IL-15rα. In some embodiments, the soluble human IL-15 ra includes amino acids 1 to 180, 1 to 181, or 1 to 182 of human IL-15 ra.

In some embodiments, the immunomodulator is IL-15SA comprising a complex of human IL-15 and a soluble human IL-15Rα comprising a truncated form of an extracellular domain that retains IL-15 binding activity and comprises a sushi domain. In the art, the sushi domain of IL-15Rα is described as being about 60 amino acids in length and comprising 4 cysteines (Wei et al, 2001). A truncated form of soluble human IL-15Rα that retains IL-15 activity and comprises a sushi domain is suitable for use in the IL-15SA of the present disclosure.

In some embodiments, immunomodulators include complexes comprising soluble human IL-15 ra described herein expressed as fusion proteins (e.g., fc fusion (e.g., human IgG1 Fc) described herein) with IL-15. In some embodiments, IL-15SA comprises a complex of a dimeric human IL-15RαFc fusion protein (e.g., human IgG1 Fc) with two human IL-15 molecules.

In some embodiments, the immunomodulator is an IL-15SA cytokine complex comprising an IL-15 molecule comprising the amino acid sequence of SEQ ID NO: 258. SEQ ID NO: 261. SEQ ID NO:262 or SEQ ID NO:263, and a polypeptide sequence as set forth in seq id no. In some embodiments, the IL-15SA cytokine complex comprises a soluble IL-15 ra molecule comprising the amino acid sequence of SEQ ID NO: 260. SEQ ID NO:264 or SEQ ID NO: 265.

In some embodiments, the immunomodulator is an IL-15SA cytokine complex comprising a complex of a dimeric IL-15RαFc fusion protein with two IL-15 molecules. In some embodiments, IL-15-SA comprises dimeric IL-15RαSu (sushi domain)/Fc (SEQ ID NO: 259) and two IL-15N72D molecules (SEQ ID NO: 258) (also referred to as ALT-803), as described in US20140134128, which is incorporated herein by reference. In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 259) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 259) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 259) and two IL-15 molecules (SEQ ID NO: 263).

In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 259) and two polypeptides having a sequence selected from the group consisting of SEQ ID NO: 258. 258, 262 and 263.

In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 258). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 263).

In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 258). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 261).

In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 258). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 263).

In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc (SEQ ID NO: 269) molecule and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc (SEQ ID NO: 259) molecule and two IL-15 molecules (SEQ ID NO: 263).

In some embodiments, IL-15SA comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260. in some embodiments, IL-15SA comprises the amino acid sequence of SEQ ID NO:261 or SEQ ID NO:262. in some embodiments, IL-15SA comprises the amino acid sequence of SEQ ID NO:261 and SEQ ID NO:259. in some embodiments, IL-15SA comprises the amino acid sequence of SEQ ID NO:262 and SEQ ID NO:259. in some embodiments, IL-15SA comprises the amino acid sequence of SEQ ID NO:263 and SEQ ID NO:259. in some embodiments, IL-15SA comprises the amino acid sequence of SEQ ID NO:261 and SEQ ID NO:260. in some embodiments, IL-15SA comprises the amino acid sequence of SEQ ID NO:262 and SEQ ID NO:260.

In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-15, or a biologically active variant thereof. Exemplary fusion proteins comprising IL-15 are depicted in fig. 36 and 37 and tables 58 and 59.

In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-15, wherein the nucleic acid is operably linked to an NFAT promoter, as described herein. Exemplary NFAT promoter-driven constructs for expression of immunomodulatory fusion proteins comprising IL-15 are depicted in table 59.

2.IL-12

In some embodiments, the modified TIL is associated with IL-12 or a variant thereof. In exemplary embodiments, IL-12 as described herein as the immunomodulatory fusion protein (e.g., membrane anchored immunomodulatory fusion protein) part.

As used herein, "interleukin 12", "IL-12" and "IL12" each refer to an interleukin that is a heterodimeric cytokine encoded by the IL-12A and IL-12B genes (Genbank accession numbers NM-000882 (IL-12A) and NM-002187 (IL-12B)). IL-12 is composed of a bundle of four alpha helices and is involved in the differentiation of naive T cells into TH1 cells. It is encoded by two independent genes, IL-12A (p 35) and IL-12B (p 40). Active heterodimers (called p 70) and p40 homodimers are formed after protein synthesis. IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12R- β1 and IL-12R- β2. IL-12 is known as a T cell stimulating factor, which stimulates the growth and function of T cells. In particular, IL-12 can stimulate production of interferon gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) from T cells and Natural Killer (NK) cells and reduce IL-4 mediated IFN-gamma inhibition. IL-12 can also mediate enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. In addition, IL-12 can also have anti-angiogenic activity by increasing the production of interferon gamma, which in turn increases the production of chemokine-induced protein-10 (IP-10 or CXCL 10), which IP-10 then mediates. Thus, without being bound by any particular theory of operation, it is believed that IL-12 may increase the survival and/or anti-tumor effects of the TIL compositions provided herein.

In some embodiments, IL-12 associated with the modified TIL is full-length IL-12, a fragment or variant of IL-12. In some embodiments, IL-12 is human IL-12 or IL-12 variants. In exemplary embodiments, IL-12 is a biologically active human IL-12 variant. In some embodiments, IL-12 and wild type IL-12 compared to 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations.

In some embodiments, IL-12 included in the modified TIL compositions comprises an IL-12p35 subunit or variant thereof. In some embodiments, IL-12p35 subunit is human IL-12p35 subunit. In some embodiments, IL-12p35 subunit has an amino acid sequence. In certain embodiments, IL-12 included in the modified TIL compositions comprises an IL-12p40 subunit or variant thereof. In certain embodiments, IL-12 is a single chain IL-12 polypeptide, which comprises an IL-12p35 subunit linked to an IL-12p40 subunit. Such IL-12 single chain polypeptides advantageously retain more than one biological activity of wild-type IL-12. In some embodiments, the single chain IL-12 polypeptides described herein from N-terminus to C-terminus according to formulas (p 40) - (L) - (p 35), where "p40" is the IL-12p40 subunit, "p35" is the IL-12p35 subunit, and L is a linker. In other embodiments, single chain IL-12 from N terminal to C terminal according to formulas (p 35) - (L) - (p 40). The single chain IL-12 polypeptide can use any suitable linker, including the linkers described herein. Suitable linkers may include, for example, linkers having the amino acid sequence (GGGGS) _x, where x is an integer from 1 to 10. Other suitable linkers include, for example, amino acid sequences GGGGGGS. Exemplary single chain IL-12 linkers that can be used with the single chain IL-12 polypeptides of the invention are also described in Lieschke et al, nature Biotechnology (Nature Biotechnology) 15:35-40 (1997), which is incorporated herein by reference in its entirety, especially with respect to the teachings of IL-12 polypeptide linkers. In exemplary embodiments, single chain IL-12 polypeptide is a single chain human IL-12 polypeptide (i.e., it contains a human p35 subunit and a p40 IL-12 subunit).

Table 6: related sequences of IL-12

In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-12, or a biologically active variant thereof.

In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-12, wherein the nucleic acid is operably linked to an NFAT promoter, as described herein. See, e.g., U.S. patent No. 8,556,882, which is incorporated herein by reference in its entirety (relevant portion relating particularly to NFAT promoters for IL-12 expression). Exemplary fusion proteins comprising IL-12 are depicted in FIGS. 36 and 37 and Table 58.

3.IL-18

In some embodiments, the modified TIL is associated with IL-18 or a variant thereof. In exemplary embodiments, IL-18 is included as part of an immunomodulatory fusion protein as described herein (e.g., a membrane anchored immunomodulatory fusion protein).

As used herein, "interleukin 18," "IL-18," "IL18," "IGIF," "IL-1g," "interferon-gamma inducing factor," and "IL1F4" each refer to an interleukin that is a heterodimeric cytokine encoded by the IL-18 gene (e.g., genbank accession numbers: NM-001243211, NM-001562, and NM-001386420). IL-18 (similar in structure to IL-1β) is a member of the IL-1 superfamily of cytokines. This cytokine is expressed by many human lymphoid and non-lymphoid cells and plays an important role in the inflammatory process. The combination of IL-18 with IL-12 activates cytotoxic T Cells (CTL) and Natural Killer (NK) cells to produce IFN-gamma, thus contributing to tumor immunity. Thus, without being bound by any particular theory of operation, it is believed that IL-18 may enhance the anti-tumor effect of the TIL compositions provided herein.

In some embodiments, the IL-18 associated with the modified TIL is full-length IL-18, a fragment or variant of IL-18. In some embodiments, IL-18 is human IL-18 or variant human IL-18. In exemplary embodiments, IL-18 is a biologically active human IL-18 variant. In some embodiments, IL-18 and wild type IL-18 compared to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations. In some embodiments, variant IL-18 has the amino acid sequence:

table 7: IL-18 related sequences

In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-18, or a biologically active variant thereof. An exemplary fusion protein comprising IL-18 is depicted in FIG. 36.

In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-18, wherein the nucleic acid is operably linked to an NFAT promoter, as described herein. Exemplary NFAT promoter-driven constructs for expression of immunomodulatory fusion proteins comprising IL-21 are depicted in table 59.

4.IL-21

In some embodiments, the modified TIL is associated with IL-21 or a variant thereof. In exemplary embodiments, IL-21 is included as part of an immunomodulatory fusion protein as described herein (e.g., a membrane anchored immunomodulatory fusion protein).

In certain embodiments, the cytokine-ABD comprises an IL-21 molecule or fragment thereof. As used herein, "interleukin 21," "IL-21," and "IL21" (e.g., genbank accession numbers: nm_001207006 and np_001193935 (human), and nm_0001291041 and np_001277970 (mouse)) each refer to members of a cytokine that bind to the IL-21 receptor and have potent modulating effects on cells of the immune system, including Natural Killer (NK) cells and cytotoxic cells, and bind to IL-21 receptors that can destroy virus-infected cells or cancer cells. Thus, without being bound by any particular theory of operation, it is believed that IL-21 may increase the survival and/or anti-tumor effects of the TIL compositions provided herein.

In some embodiments, IL-21 is human IL-21. In some embodiments, the IL-21 associated with the modified TIL is full-length IL-21, a fragment or variant of IL-21. In some embodiments, IL-21 is human IL-21 or a variant of human IL-21. In exemplary embodiments, IL-21 is a biologically active human IL-21 variant. In some embodiments, IL-21 and wild type IL-21 compared to 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations.

Table 8: related sequences of IL-21.

In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-21 or a biologically active variant thereof. Exemplary fusion proteins comprising IL-21 are depicted in FIGS. 36 and 37 and tables 58 and 59.

In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-21, wherein the nucleic acid is operably linked to an NFAT promoter, as described herein.

5.IL-2

In some embodiments, the modified TIL is associated with IL-2 or a variant thereof. In exemplary embodiments, IL-2 is included as part of an immunomodulatory fusion protein as described herein (e.g., a membrane anchored immunomodulatory fusion protein).

In certain embodiments, the cytokine-ABD comprises an IL-2 molecule or fragment thereof. As used herein, "interleukin 2", "IL-2", "IL2" and "TCGF" (e.g., genbank accession numbers: NM-000586 and NP-000577 (human)) all refer to members of the cytokines that bind the IL-2 receptor. IL-2 enhances activation-induced cell death (AICD). IL-2 also promotes T-cell differentiation into effector T-cells and memory T-cells when the naive T-cells are also stimulated by antigen, thereby helping the body to fight infection. IL-2, along with other cytokines, stimulates the differentiation of naive CD4+ T cells into Th1 and Th2 lymphocytes and prevents differentiation into Th17 and follicular Th lymphocytes. IL-2 also increases the cell killing activity of natural killer cells and cytotoxic T cells. Thus, without being bound by any particular theory of operation, it is believed that IL-2 may increase the survival and/or anti-tumor effects of the TIL compositions provided herein.

In some embodiments, IL-2 is human IL-2. In some embodiments, the IL-2 associated with the modified TIL is full length IL-2, a fragment or variant of IL-2. In some embodiments, IL-2 is human IL-2 or a variant of human IL-2. In exemplary embodiments, IL-2 is a biologically active human IL-2 variant. In some embodiments, IL-2 and wild type IL-2 compared to 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations.

Table 9: related sequences of IL-2

In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-2, or a biologically active variant thereof. Exemplary fusion proteins comprising IL-2 are depicted in FIGS. 36 and 37.

In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-2, wherein the nucleic acid is operably linked to an NFAT promoter, as described herein.

CD40 agonists

In some embodiments, the modified TIL binds to a CD40 agonist. In exemplary embodiments, a CD40 agonist is included as part of an immunomodulatory fusion protein as described herein (e.g., a membrane anchored immunomodulatory fusion protein).

Cluster of differentiation 40 (i.e., CD 40) is a costimulatory protein found on Antigen Presenting Cells (APCs) and is required for APC activation. Binding of CD40L (CD 154) to CD40 on T helper cells can activate antigen presenting cells (e.g., dendritic cells) and induce various downstream effects. Without being bound by any particular theory of operation, it is believed that the addition of more than one immunomodulator that activates CD40 on antigen presenting cells (i.e., CD40 agonist) may enhance the anti-tumor effect of the TIL compositions provided herein. CD40 agonists include, for example, CD40L, which agonistically binds CD40, and antibodies or antibody fragments thereof (e.g., scFV). In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising CD40L or a biologically active variant thereof. In some embodiments, the TIL compositions comprise an immunomodulatory fusion protein comprising an agonistic anti-CD 40 binding domain (e.g., scFv). The sequences of exemplary CD40 agonists are depicted in the table below.

The activity of a CD40 agonist may be measured using any suitable method known in the art. For example, engagement (ligation) of CD40 on DCs can induce increased costimulatory and MHC molecule surface expression, pro-inflammatory cytokine production, and enhanced T cell triggering. CD40 engagement on resting B cells can increase antigen presenting function and proliferation. In exemplary embodiments, the CD40 agonist is capable of activating human dendritic cells.

In some embodiments, the TIL compositions comprise an agonistic anti-CD 40 binding domain having VH and VL sequences of an anti-CD 40 scFv depicted in table 10, or biologically active variants thereof. In some embodiments, the anti-CD 40 binding domain comprises a VH sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a VH sequence depicted in table 10. In some embodiments, the agonistic anti-CD 40 binding domain comprises a VH sequence comprising 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the VH sequence depicted in table 10. In some embodiments, the anti-CD 40 binding domain comprises a VL sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a VL sequence depicted in table 10. In some embodiments, the anti-CD 40 binding domain comprises a VL sequence comprising 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the VL sequence depicted in table 10. In an exemplary embodiment, the anti-CD 40 binding domain is a sequence selected from the group consisting of SEQ ID NOs: 276. 279, 282 and 285 are anti-CD 40 scFv.

In some embodiments, the anti-CD 40 binding domain is a variant of the anti-CD 40 scFv in table 10 that is capable of binding to human CD40. In an exemplary embodiment, the variant of the anti-CD 40 scFv hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 276. 279, 282 and 285 have at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity.

Assessment of CD40 binding domain binding can be measured using any suitable assay known in the art, including (but not limited to): biacore, surface Plasmon Resonance (SPR) and/or BLI (biolayer interferometry, e.g., octet assay) assays.

Other CD40 binding domains (VH and VL) suitable for use as immunomodulators include the CD40 binding domains described in U.S. Pat. nos. 6,838,261, US6,843,989, US 7,338,660, US 8,7778,345, which are incorporated herein by reference, especially with respect to the teachings of anti-CD 40 antibodies and VH, VL and CDR sequences.

In some embodiments, the CD40 agonist is a CD40 ligand (CD 40L). In an exemplary embodiment, CD40L is human CD40L (SEQ ID NO: 270). In some embodiments, CD40L is a variant of human CD40L that hybridizes to SEQ ID NO:253 has at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, CD40L is a variant of human CD40L that hybridizes to SEQ ID NO:273 comprises 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to 273.

Exemplary fusion proteins comprising CD40 agonists are depicted in fig. 36 and 37.

In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising a CD40 agonist, wherein the nucleic acid is operably linked to an NFAT promoter, as described herein.

Table 10: related sequences of CD40 agonists

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IV. Gene editing Process

A. overview: TIL amplification+Gene editing+transient Gene editing

In some embodiments of the present invention, the methods for amplifying a population of TILs comprise one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic effect. As used herein, "gene editing," "editing of a gene," and "genome editing" refer to a genetic modification in which DNA is permanently modified in the genome of a cell, such as by insertion, deletion, modification, or substitution of DNA within the genome of a cell. In some embodiments, gene editing results in silencing (sometimes referred to as gene knockout) or inhibition/reduction (sometimes referred to as gene knockdown) of expression of the DNA sequence. In other embodiments, gene editing results in enhanced expression of the DNA sequence (e.g., by causing overexpression). According to embodiments of the present invention, gene editing techniques are used to enhance the effectiveness of therapeutic TIL populations.

The method of amplifying tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be performed according to any embodiment of the methods described herein (e.g., the exemplary TIL amplification method referred to as process 2A is described below), wherein the method further comprises gene editing at least a portion of the TILs. According to other embodiments, the method of amplifying a TIL into a therapeutic population of TILs is performed according to any of the embodiments of the methods described in U.S. patent No. 10,517,894, U.S. patent application publication No. 2020/011719 A1, or U.S. patent No. 10,894,063, which are incorporated herein by reference in their entirety, wherein the method further comprises gene editing at least a portion of the TILs. Thus, some embodiments of the invention provide a therapeutic TIL population that has been amplified according to any of the embodiments described herein, wherein at least a portion of the therapeutic population has been gene-edited, e.g., at least a portion of the therapeutic TIL population transferred to an infusion bag has been permanently gene-edited.

In some embodiments of the invention regarding methods of amplifying a population of TILs, the methods include one or more steps of introducing a nucleic acid (e.g., mRNA) for transient expression of an immunomodulatory protein (e.g., an immunomodulatory fusion protein comprising an immunomodulatory protein fused to a membrane anchor) into at least a portion of a TIL to produce a modified TIL having (i) reduced dependence on cytokines when amplified in culture, and/or (ii) enhanced therapeutic effects. As used herein, "transient gene editing", "transient phenotype change", "transient phenotype modification", "transient cell change", "transient cell modification", "transient expression", "transient change in protein expression", a transient cell change in expression, a transient change in expression of a protein, a transient change in a protein, a transient cell change, a protein, a transient cell change, a protein, a "transient modification", "transient phenotypic change", "non-permanent phenotypic change", "transient modification", "non-permanent modification", "transient change", grammatical variations of any of the foregoing and any expression having similar meanings refer to a cellular modification or phenotypic change, wherein a nucleic acid (e.g., mRNA) is introduced into a cell, the nucleic acid is transferred into the cell, e.g., by electroporation, calcium phosphate transfection, viral transduction, etc., and expressed in the cell (e.g., expression of an immunomodulatory protein (e.g., an immunomodulatory fusion protein comprising an immunomodulatory protein fused to a membrane anchor) to effect a transient or non-permanent phenotypic change in the cell, e.g., transient display of the membrane-anchored immunomodulatory fusion protein on the cell surface. According to embodiments of the invention, transient phenotype altering techniques are used to reduce the dependence on cytokines in culture upon TIL expansion and/or to enhance the effectiveness of therapeutic TIL populations.

In some embodiments, intracellular delivery of nucleic acids encoding fusion proteins provided herein is performed using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. The SQZ platform is capable of delivering nucleic acids and proteins to a variety of primary human cells, including T cells (Sharei et al, proc. Natl. Acad. Sci. USA (PNAS) 2013, and Sharei et al, sci. Public library complex (PLOS ONE) 2015 and Greisbeck et al, J.Immunol 195, 2015). In the SQZ platform, delivery of nucleic acids encoding immunomodulatory fusion proteins into cells is achieved by the microfluidic contraction to temporarily disrupt the cell membrane of the cell (e.g., TIL) for modification. Such methods as described in international patent application publication No. WO 2013/059343A1, WO 2017/008063A1 or WO 2017/123663A1 or U.S. patent application publication No. US2014/0287509A1, US2018/0201889A1 or US2018/0245089A1 may be used in the present invention to deliver nucleic acids encoding immunomodulatory fusion proteins of the invention to a TIL population. In some embodiments, the delivered nucleic acid allows transient protein expression of the immunomodulatory fusion protein in the modified TIL. In some embodiments, the delivered nucleic acid encoding the immunomodulatory fusion protein is stably incorporated into the TIL cell genome using the SQZ platform.

B. Time of gene editing/transient phenotype changes during TIL amplification

According to some embodiments, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises:

(a) Obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) Adding tumor fragments into the closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and optionally OKT-3 (e.g., OKT-3 may be present in the culture medium starting at the start of the amplification process), producing a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area for about 3 to 14 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(d) Performing a second amplification by supplementing the cell culture medium of a second population of TILs with additional IL-2, optional OKT-3, and Antigen Presenting Cells (APC), resulting in a third population of TILs, wherein the second amplification is performed for about 7 to 14 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(G) At any time during the method of step (f) prior to transfer to the infusion bag, at least a portion of the TIL cells are genetically edited to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells. In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

As described in step (g) of the embodiments described above, the gene editing process may be performed at any time during the TIL amplification method before transfer to the infusion bag in step (f), which means that the TIL may be gene edited before, during or after any of the amplification methods, e.g., during any of steps (a) to (f) outlined in the methods above or before or after any of steps (a) to (e) outlined in the methods above. According to certain embodiments, TILs are collected during the amplification method (e.g., the amplification method that "pauses" at least a portion of the TILs) and subjected to a gene editing process, in some cases, then back into the amplification method (e.g., back into the culture medium) to continue the amplification process, such that the therapeutic population of TILs that is ultimately transferred to the infusion bag is permanently gene edited. In some embodiments, the gene editing process may be performed prior to amplification by activating the TIL, performing a gene editing step on the activated TIL, and amplifying the gene-edited TIL according to the methods described herein. In some embodiments, the nucleic acid for gene editing is delivered to the TIL using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

In some embodiments, the first TIL amplification step is followed by a gene editing process. In some embodiments, the gene editing process is performed after the first TIL amplification step and before the second amplification step. In some embodiments, the gene editing process is performed after TIL activation. In some embodiments, the gene editing process is performed after the first amplification step and after TIL activation, but before the second amplification step. In some embodiments, the gene editing process is performed after the first amplification step and after the TIL is activated, and the TIL is left to stand after the gene editing and before the second amplification step. In some embodiments, the TIL is left to stand for about 1 to 2 days after gene editing and before the second amplification step. In some embodiments, TIL is activated by exposure to an anti-CD 3 agonist as well as an anti-CD 28 agonist. In some embodiments, the anti-CD 3 agonist is an anti-CD 3 agonist antibody and the anti-CD 28 agonist is an anti-CD 28 agonist antibody. In some embodiments, the anti-CD 3 agonist antibody is OKT-3. In some embodiments, the TIL is activated by exposure to anti-CD 3 agonist antibodies and anti-CD 28 agonist antibody conjugated beads. In some embodiments, the anti-CD 3 agonist antibody and anti-CD 28 agonist antibody conjugated beads are the TransAct ^TM product of Miltenyi. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction. In some embodiments, the gene editing process is performed by lentiviral transduction. In some embodiments, the immunomodulatory composition is a membrane anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21. In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises: a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TIL is genetically edited to express an immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express a first immunomodulatory fusion protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter.

In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction. In some embodiments, the gene editing process is performed by lentiviral transduction.

(b) Adding tumor fragments into the closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and optionally OKT-3 (e.g., OKT-3 may be present in the culture medium starting at the start of the amplification process), producing a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area, the first amplification being performed for about 3 to 14 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(d) Gene editing is performed on at least a portion of the TIL cells in the second population of TIL cells, and an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) is expressed on the surface of the TIL cells.

(E) Performing a second amplification by supplementing the cell culture medium of a second population of TILs with additional IL-2, optional OKT-3, and Antigen Presenting Cells (APC), resulting in a third population of TILs, wherein the second amplification is performed for about 7 to 14 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(f) Collecting the population of therapeutic TILs obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system;

(g) Transferring the collected TIL population from step (e) to an infusion bag, wherein the transferring of steps (e) to (f) is performed without opening the system. In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the TIL is left to stand after the gene editing step and before the second amplification step. In some embodiments, the TIL is left to stand for about 1 to 2 days after the gene editing step and before the second amplification step. In some embodiments, TIL is activated by exposure to an anti-CD 3 agonist and an anti-CD 28 agonist. In some embodiments, the anti-CD 3 agonist is an anti-CD 3 agonist antibody and the anti-CD 28 agonist is an anti-CD 28 agonist antibody. In some embodiments, the anti-CD 3 agonist antibody is OKT-3. In some embodiments, the TIL is activated by exposure to anti-CD 3 agonist antibodies and anti-CD 28 agonist antibody conjugated beads. In some embodiments, the anti-CD 3 agonist antibody and anti-CD 28 agonist antibody conjugated beads are the TransAct ^TM product of Miltenyi. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction of TIL, optionally for about 2 days. In some embodiments, the gene editing process is performed by lentiviral transduction of the TIL, optionally for about 2 days. In some embodiments, the immunomodulatory composition is a membrane anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21. In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises: a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TIL is genetically edited to express an immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express a first immunomodulatory fusion protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter.

It should be noted that alternative embodiments of the amplification process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a) through (g), and may have a different number of steps. Regardless of the particular embodiment, the gene editing process may be performed at any time during the TIL amplification method. For example, alternative embodiments may include more than two amplifications, the TIL may be gene edited during a third or fourth amplification, etc.

According to other embodiments, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises:

(b) Adding tumor fragments into the closed system;

(G) Introducing a transient phenotypic change into at least a portion of the TIL cells at any time during the method of step (f) prior to transfer to the infusion bag to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells. In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the nucleic acid for transient phenotypic change is delivered to the TIL using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

As described in step (g) of the embodiments described above, the transient phenotype change process may be performed at any time during the TIL amplification method prior to transferring the infusion bag in step (f), which means that the transient phenotype change may be performed on the TIL before, during or after any of the amplification methods, e.g., during any of steps (a) to (f) outlined in the methods above or before or after any of steps (a) to (e) outlined in the methods above. According to certain embodiments, TILs are collected during the amplification method (e.g., the amplification method that "pauses" at least a portion of the TILs) and subjected to a transient modification process, in some cases, then back into the amplification method (e.g., back into the medium) to continue the amplification process, such that at least a portion of the therapeutic TIL population eventually transferred into the infusion bag is transiently altered to express the immunomodulatory composition on the surface of the TIL cells. In some embodiments, the transient cell modification process may be performed prior to expansion by activating the TIL, performing a transient phenotype modification step on the activated TIL, and expanding the modified TIL according to the methods described herein.

It should be noted that alternative embodiments of the amplification process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a) through (g), and may have a different number of steps. Regardless of the particular embodiment, the transient cell modification process may be performed at any time during the TIL amplification method. For example, alternative embodiments may include more than two amplifications, the TIL may be subjected to a transient cell modification process during a third or fourth amplification, etc.

According to some embodiments, the TIL from one or more of the first population, the second population, and the third population is subjected to a gene editing process. For example, a first population of TILs or a portion of the collected TILs from the first population may be subjected to gene editing, after which these TILs may then be placed back into the amplification process (e.g., back into the culture medium). Alternatively, the TILs from the second or third population, or a portion of the TILs collected from the second or third population, respectively, may be subjected to gene editing, after which these TILs may then be placed back into the amplification process (e.g., back into the culture medium). According to other embodiments, gene editing is performed while the TIL is still in the medium and while amplification is in progress, i.e., without "removing" the TIL from the amplification.

According to some embodiments, transient cell modification processes are performed on TILs from one or more of the first population, the second population, and the third population. For example, a first population of TILs or a portion of the collected TILs from the first population may be subjected to transient cell modifications, and after the gene editing process, these transient modified TILs may then be placed back into the amplification process (e.g., back into the culture medium). Alternatively, transient cell modifications may be made to TILs from the second or third populations or a portion of the collected TILs from the second or third populations, respectively, and after the transient cell modification process, these modified TILs may then be placed back into the amplification process (e.g., back into the culture medium). According to other embodiments, transient cell modification is performed while the TIL is still in the medium and while the amplification is in progress, i.e., transient cell modification is achieved without "removing" the TIL from the amplification.

According to other embodiments, the TIL from the first amplification or the TIL from the second amplification, or both, are subjected to a gene editing process. For example, during the first amplification or the second amplification, TILs collected from the culture medium may be subjected to gene editing, after which these TILs may then be put back into the amplification method, for example by reintroducing them back into the culture medium.

According to other embodiments, transient cell modification processes are performed on the TIL from the first amplification or the TIL from the second amplification, or both. For example, during the first amplification or the second amplification, the TILs collected from the culture medium may be subjected to transient cell modifications, after which these modified TILs may then be put back into the amplification method, for example by reintroducing them back into the culture medium.

According to other embodiments, at least a portion of the TIL is subjected to a gene editing process after the first amplification and before the second amplification. For example, after a first amplification, TILs collected from the culture medium may be subjected to gene editing, after which these TILs may then be put back into the amplification method (e.g., by reintroducing them back into the culture medium) for a second amplification.

According to other embodiments, at least a portion of the TIL is subjected to a transient cell modification process after the first amplification and before the second amplification. For example, after a first amplification, the TILs collected from the culture medium may be subjected to transient cell modifications, after which these modified TILs may then be put back into the amplification method (e.g., by reintroducing them back into the culture medium) for a second amplification.

According to alternative embodiments, the gene editing process is performed before step (c) (e.g., before, during or after any of steps (a) to (b)), before step (d) (e.g., before, during or after any of steps (a) to (c)), before step (e) (e.g., before, during or after any of steps (a) to (d)), or before step (f) (e.g., before, during or after any of steps (a) to (e)).

According to alternative embodiments, the transient cell modification process is performed before step (c) (e.g., before, during or after any of steps (a) to (b)), before step (d) (e.g., before, during or after any of steps (a) to (c)), before step (e) (e.g., before, during or after any of steps (a) to (d)), or before step (f) (e.g., before, during or after any of steps (a) to (e)).

It should be noted that with respect to OKT-3, according to certain embodiments, the cell culture medium may comprise OKT-3 starting on the starting day (day 0) or day 1 of the first amplification, such that on day 0 and/or day 1, the TIL is subjected to gene editing or transient cell modification after it has been exposed to OKT-3 in the cell culture medium. According to other embodiments, the cell culture medium comprises OKT-3 during the first amplification and/or during the second amplification, and the gene editing or transient cell modification is performed prior to introducing OKT-3 into the cell culture medium. Alternatively, the cell culture medium may comprise OKT-3 during the first amplification and/or during the second amplification, the gene editing or transient cell modification being performed after the OKT-3 is introduced into the cell culture medium.

It should also be noted that with respect to the 4-1BB agonist, according to certain embodiments, the cell culture medium may comprise the 4-1BB agonist starting on the starting day of the first amplification (day 0) or day 1, such that on day 0 and/or day 1, the TIL is subjected to gene editing or transient cell modification after it has been exposed to the 4-1BB agonist in the cell culture medium. According to other embodiments, the cell culture medium comprises a 4-1BB agonist during the first amplification and/or during the second amplification, and the gene editing or transient cell modification is performed prior to introducing the 4-1BB agonist into the cell culture medium. Alternatively, the cell culture medium may comprise 4-1BB during the first amplification and/or during the second amplification, and the gene editing or transient cell modification may be performed after introducing the 4-1BB into the cell culture medium.

It should also be noted that with respect to IL-2, according to certain embodiments, the cell culture medium may comprise IL-2 starting on the starting day of the first amplification (day 0) or day 1, such that on day 0 and/or day 1, the TIL is subjected to gene editing or transient cell modification after it has been exposed to IL-2 in the cell culture medium. According to other embodiments, the cell culture medium comprises IL-2 during the first amplification and/or during the second amplification, and the gene editing or transient cell modification is performed prior to introducing IL-2 into the cell culture medium. Alternatively, the cell culture medium may comprise IL-2 during the first amplification and/or during the second amplification, with gene editing or transient cell modification following introduction of IL-2 into the cell culture medium.

As described above, the cell culture medium may comprise one or more of OKT-3, 4-1BB agonist and IL-2 starting on day 0 or day 1 of the first amplification. According to some embodiments, the cell culture medium comprises OKT-3 starting on day 0 or day 1 of the first amplification, and/or the cell culture medium comprises 4-1BB agonist starting on day 0 or day 1 of the first amplification, and/or the cell culture medium comprises IL-2 starting on day 0 or day 1 of the first amplification. According to other examples, the cell culture medium comprises OKT-3 and 4-1BB agonists starting on day 0 or day 1 of the first expansion. According to other examples, the cell culture medium comprises OKT-3, a 4-1BB agonist and IL-2 starting on day 0 or day 1 of the first expansion. Of course, more than one of OKT-3, 4-1BB agonist and IL-2 may be added to the cell culture medium at more than one other point in time during the amplification process, as described in the various embodiments described herein.

(b) Adding tumor fragments into the closed system;

(c) Performing a first amplification by culturing the first population of TILs in a cell culture medium comprising IL-2 for about 3 to 11 days, producing a second population of TILs, wherein the first amplification is performed in a closed container providing a first gas permeable surface area;

(d) Activating a second population of TILs by adding OKT-3 and incubating for about 1 to 2 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Gene editing at least a portion of the TIL cells in the second population of TILs to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells;

(f) Optionally, the second TIL population is allowed to stand for about 1 day;

(g) Performing a second amplification by supplementing a cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and Antigen Presenting Cells (APC), producing a third TIL population, wherein the second amplification is performed for about 7 to 11 days to obtain the third TIL population, wherein the second amplification is performed in a closed container providing a second gas permeable surface area, the transition from step (f) to step (g) being performed without opening the system;

(h) Collecting the therapeutic TIL population obtained from step (g) to obtain a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, the collected TIL population being a therapeutic TIL population; and

(I) Transferring the collected TIL population to an infusion bag, wherein the transfer from step (h) to (i) is performed without opening the system. In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the TIL is left to stand after the gene editing step and before the second amplification step. In some embodiments, the TIL is left to stand for about 1 to 2 days after the gene editing step and before the second amplification step. In some embodiments, TIL is activated by exposure to an anti-CD 3 agonist and an anti-CD 28 agonist for about 2 days. In some embodiments, the anti-CD 3 agonist is an anti-CD 3 agonist antibody and the anti-CD 28 agonist is an anti-CD 28 agonist antibody. In some embodiments, the anti-CD 3 agonist antibody is OKT-3. In some embodiments, the TIL is activated by exposure to anti-CD 3 agonist antibodies and anti-CD 28 agonist antibody conjugated beads. In some embodiments, the anti-CD 3 agonist antibody and anti-CD 28 agonist antibody conjugated beads are the TransAct ^TM product of Miltenyi. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction of TIL, optionally for about 2 days. In some embodiments, the gene editing process is performed by lentiviral transduction of the TIL, optionally for about 2 days. In some embodiments, the immunomodulatory composition is a membrane anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21. In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises: a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TIL is genetically edited to express an immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express a first immunomodulatory fusion protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter.

(b) Adding tumor fragments into the closed system;

(c) Performing a first amplification by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days, producing a second population of TILs, wherein the first amplification is performed in a closed container providing a first gas permeable surface area;

(d) Stimulating a second TIL population by adding OKT-3 and incubating for about 1 to 3 days, wherein the transition from step (c) to step (d) is performed without opening the system;

(e) Performing sterile electroporation of the second population of TILs to effect transfer of the at least one gene editor into a portion of cells of the second population of TILs;

(f) Standing the second TIL population for about 1 day;

(g) Performing a second amplification by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and Antigen Presenting Cells (APC), producing a third TIL population, wherein the second amplification is performed for about 7 to 11 days to obtain the third TIL population, performing the second amplification in a closed container providing a second gas permeable surface area, the transition from step (f) to step (g) being performed without opening the system;

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

Wherein at least one gene editor (editor) is aseptically electroporated into a portion of the cells of the second TIL population to modify a plurality of cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(e) Performing sterile electroporation of the second population of TILs to effect transfer of the at least one nucleic acid molecule into a portion of cells of the second population of TILs;

(f) Standing the second TIL population for about 1 day;

Wherein sterile electroporation of at least one nucleic acid molecule into a portion of cells of the second TIL population modifies a plurality of cells in the portion to transiently express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein at least one gene editor is aseptically electroporated into a portion of the cells of the second TIL population to modify a plurality of cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(e) Temporarily disrupting the cell membrane of the second TIL population to effect transfer of the at least one gene editor into a portion of the cells of the second TIL population;

(f) Standing the second TIL population for about 1 day;

(h) Collecting the therapeutic TIL population obtained from step (g) to obtain a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population; and

Wherein at least one gene editor delivered to a portion of cells of the second TIL population modifies a plurality of cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cell membrane of the second TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

(b) Adding tumor fragments into the closed system;

(e) Temporarily disrupting the cell membrane of the second TIL population to effect transfer of the at least one nucleic acid molecule into a portion of the cells of the second TIL population;

(f) Standing the second TIL population for about 1 day;

(g) Performing a second amplification by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and Antigen Presenting Cells (APC), producing a third TIL population, wherein the second amplification is performed for about 7 to 11 days to obtain the third TIL population, and the transition from step (f) to step (g) is performed without opening the system in a closed container providing a second gas permeable surface area;

Wherein at least one nucleic acid molecule delivered to a portion of cells of the second TIL population modifies a plurality of cells in the portion to transiently express the immunomodulatory composition on the cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cell membrane of the second TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

In some embodiments, provided herein are methods of preparing expanded tumor-infiltrating lymphocytes (TILs), comprising:

(a) Obtaining and/or receiving a first population of TILs from tumor tissue resected from a subject or patient;

(b) Culturing the first population of TILs in a first cell culture medium comprising IL-2 for about 3 to 9 days, resulting in a second population of TILs;

(c) Activating the second TIL population with anti-CD 3 and anti-CD 28 beads or antibodies for 1 to 7 days, resulting in a third TIL population;

(d) Performing sterile electroporation on the third population of TILs to effect transfer of the at least one gene editor into a portion of the cells of the third population of TILs, resulting in a fourth population of TILs; and

(E) Culturing the fourth population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3 and IL-2 for about 5 to 15 days, producing an amplified number of TILs,

Wherein at least one gene editor is aseptically electroporated into a portion of the cells of the third TIL population to modify a plurality of the cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(d) Gene editing at least a portion of the TIL cells in the second population of TILs to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells; and

(E) Culturing the fourth population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3, and IL-2 for about 5 to 15 days, resulting in an expanded number of TILs.

In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the TIL is left to stand after the gene editing step and before the second amplification step. In some embodiments, the TIL is left to stand for about 1 to 2 days after the gene editing step and before the second amplification step. In some embodiments, TIL is activated by exposure to an anti-CD 3 agonist and an anti-CD 28 agonist for about 2 days. In some embodiments, the anti-CD 3 agonist is an anti-CD 3 agonist antibody and the anti-CD 28 agonist is an anti-CD 28 agonist antibody. In some embodiments, the anti-CD 3 agonist antibody is OKT-3. In some embodiments, the TIL is activated by exposure to anti-CD 3 agonist antibodies and anti-CD 28 agonist antibody conjugated beads. In some embodiments, the anti-CD 3 agonist antibody and anti-CD 28 agonist antibody conjugated beads are the TransAct ^TM product of Miltenyi. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction of TIL, optionally for about 2 days. In some embodiments, the gene editing process is performed by lentiviral transduction of the TIL, optionally for about 2 days. In some embodiments, the immunomodulatory composition is a membrane anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21. In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises: a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TIL is genetically edited to express an immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TIL is genetically edited to express a first immunomodulatory fusion protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter.

(d) Performing sterile electroporation of the third population of TILs to effect transfer of the at least one nucleic acid molecule into a portion of cells of the third population of TILs, resulting in a fourth population of TILs; and

Wherein at least one nucleic acid molecule delivered to a portion of cells of the third TIL population modifies a plurality of cells in the portion to transiently express the immunomodulatory composition on the cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(c) Culturing the first population of TILs in a first cell culture medium comprising IL-2 for about 3 to 9 days, resulting in a second population of TILs;

(d) Activating the second TIL population with anti-CD 3 and anti-CD 28 beads or antibodies for 1 to 7 days, resulting in a third TIL population;

(e) Gene editing at least a portion of the TIL cells in the second population of TILs to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells; and

(F) Culturing the fourth population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3, and IL-2 for about 5 to 15 days, resulting in an expanded number of TILs.

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(e) Performing sterile electroporation on the third population of TILs to effect transfer of the at least one gene editor into a portion of the cells of the third population of TILs, resulting in a fourth population of TILs; and

(F) Culturing the fourth population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3 and IL-2 for about 5 to 15 days, producing an amplified number of TILs,

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(e) Performing sterile electroporation of the third population of TILs to effect transfer of the at least one nucleic acid molecule into a portion of cells of the third population of TILs, resulting in a fourth population of TILs; and

(d) Temporarily disrupting the cell membrane of the third TIL population to effect transfer of the at least one gene editor into a portion of the cells of the third TIL population, resulting in a fourth TIL population; and

Wherein transferring at least one gene editor into a portion of cells of the third TIL population modifies a plurality of cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cell membrane of the second TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

(d) Temporarily disrupting the cell membrane of the third TIL population to effect transfer of the at least one nucleic acid molecule into a portion of the cells of the third TIL population, producing a fourth TIL population; and

Wherein transferring at least one nucleic acid molecule into a portion of cells of the third TIL population modifies a plurality of cells in the portion to transiently express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cell membrane of the second TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(e) Temporarily disrupting the cell membrane of the third TIL population to effect transfer of the at least one gene editor into a portion of the cells of the third TIL population, resulting in a fourth TIL population; and

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(e) Temporarily disrupting the cell membrane of the third TIL population to effect transfer of the at least one nucleic acid molecule into a portion of the cells of the third TIL population, producing a fourth TIL population; and

Wherein transferring at least one nucleic acid molecule into a portion of cells of the third TIL population modulates a plurality of cells in the portion to transiently express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

In some embodiments, any of the foregoing methods is modified such that the step of culturing the fourth TIL population is replaced by the steps of:

(f) Culturing the fourth TIL population in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3, and IL-2 for about 1 to 7 days, producing a culture of a fifth TIL population; and

(G) Splitting (split) the culture of the fifth TIL population into a plurality of subcultures, culturing each of the plurality of subcultures in a third cell culture medium comprising IL-2 for about 3 to 7 days, and combining the plurality of subcultures to obtain an amplified number of TILs.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 1, 2, 3, 4, 5, 6, or 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 2 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 3 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 4 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 5 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 6 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 1 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 1 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 1 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 1 to 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 1 to 2 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 2 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 3 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 4 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 5 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 3 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 3 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 2 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 2 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 2 to 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 4 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 1 day.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 2 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population occurs for about 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population is performed for about 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population occurs for about 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of activating the second TIL population occurs for about 7 days.

In some embodiments, provided herein are methods for preparing expanded tumor-infiltrating lymphocytes (TILs), comprising:

(b) Culturing the first population of TILs in a first cell culture medium comprising IL-2 and OKT-3 for about 3 to 9 days, resulting in a second population of TILs;

(c) Performing aseptic electroporation on the second population of TILs to effect transfer of the at least one gene editor into a portion of the cells of the second population of TILs, resulting in a third population of TILs; and

(D) Culturing the third population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3 and IL-2 for about 5 to 15 days, producing an amplified number of TILs,

Wherein at least one gene editor is aseptically electroporated into a portion of the cells of the third TIL population to modify a plurality of the cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(c) Performing sterile electroporation of the second population of TILs to effect transfer of the at least one nucleic acid molecule into a portion of cells of the second population of TILs, resulting in a third population of TILs; and

Wherein sterile electroporation of at least one nucleic acid molecule into a portion of cells of the third TIL population modifies a plurality of cells in the portion to transiently express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(c) Culturing the first population of TILs in a first cell culture medium comprising IL-2 and OKT-3 for about 3 to 9 days, resulting in a second population of TILs;

(d) Performing aseptic electroporation on the second population of TILs to effect transfer of the at least one gene editor into a portion of the cells of the second population of TILs, resulting in a third population of TILs; and

(E) Culturing the third population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3 and IL-2 for about 5 to 15 days, producing an amplified number of TILs,

Wherein at least one gene editor is aseptically electroporated into a portion of the cells of the second TIL population to modify a plurality of cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the cytokine is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cytokine is selected from: IL-2, IL-12, IL-15, IL-18 and IL-21. In some embodiments, the cytokine is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(d) Performing sterile electroporation of the second population of TILs to effect transfer of the at least one nucleic acid molecule into a portion of cells of the second population of TILs, resulting in a third population of TILs; and

(c) Temporarily disrupting the cell membrane of the second population of TILs to effect transfer of the at least one gene editor into a portion of the cells of the second population of TILs, resulting in a third population of TILs; and

Wherein transferring at least one gene editor into a portion of cells of the second TIL population modifies a plurality of cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cell membrane of the second TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

(c) Temporarily disrupting the cell membrane of the second TIL population to effect transfer of the at least one nucleic acid molecule into a portion of the cells of the second TIL population, producing a third TIL population; and

Wherein transferring at least one gene editor into a portion of cells of the second TIL population modifies a plurality of cells in the portion to transiently express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cell membrane of the second TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(d) Temporarily disrupting the cell membrane of the second TIL population to effect transfer of the at least one gene editor into a portion of the cells of the second TIL population to produce a third TIL population; and

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(d) Temporarily disrupting the cell membrane of the second TIL population to effect transfer of the at least one nucleic acid molecule into a portion of the cells of the second TIL population, producing a third TIL population; and

Wherein transferring at least one nucleic acid molecule into a portion of cells of the second TIL population modifies a plurality of cells in the portion to transiently express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the cell membrane of the second TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

In some embodiments, the step of culturing the third TIL population is performed by: culturing the third population of TILs in the second cell culture medium for a first period of about 1 to 7 days, splitting the culture into a plurality of subcultures at the end of the first period, culturing each of the plurality of subcultures in the third culture medium comprising IL-2 for a second period of about 3 to 7 days, and combining the plurality of subcultures at the end of the second period to obtain an amplified number of TILs.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 4 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 5 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 6 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 7 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 8 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 9 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 10 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 4 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 5 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 6 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 7 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 8 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 9 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 4 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 5 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 6 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 7 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 8 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 3 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 3 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 3 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 3 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 3 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 4 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 4 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 4 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 5 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 5 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 5 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 6 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 6 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 7 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 4 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first population of TILs in the first cell culture medium is performed for about 11 days.

(b) Culturing the first population of TILs in a first cell culture medium comprising IL-2 for about 3 days to produce a second population of TILs;

(c) Culturing the second population of TILs in a second cell culture medium comprising IL-2 and OKT-3 for 2 to 4 days, resulting in a third population of TILs;

(E) Culturing the fourth population of TILs in a third cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3 and IL-2 for about 5 to 15 days, producing an amplified number of TILs,

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(c) Culturing the first population of TILs in a first cell culture medium comprising IL-2 for about 3 days to produce a second population of TILs;

(d) Culturing the second population of TILs in a second cell culture medium comprising IL-2 and OKT-3 for 2 to 4 days, resulting in a third population of TILs;

(F) Culturing the fourth population of TILs in a third cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3 and IL-2 for about 5-15 days to produce an amplified number of TILs,

Wherein sterile electroporation of at least one gene editor into a portion of cells of the third TIL population modulates a plurality of cells in the portion to express the immunomodulatory composition on a cell surface. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(F) Culturing the fourth population of TILs in a third cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3 and IL-2 for about 5 to 15 days, producing an amplified number of TILs,

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

In some embodiments, the step of culturing the fourth TIL population is performed by: culturing the fourth population of TILs in the third cell culture medium for a first period of about 1 to 7 days, splitting the culture into a plurality of subcultures at the end of the first period, culturing each of the plurality of subcultures in the fourth culture medium comprising IL-2 for a second period of about 3 to 7 days, and combining the plurality of subcultures at the end of the second period to obtain an amplified number of TILs.

In some embodiments, in the step of culturing the first TIL population in a first medium, the first medium further comprises anti-CD 3 and anti-CD 28 beads or antibodies.

In some embodiments, the anti-CD 3 and anti-CD 28 beads or antibodies comprise OKT-3 in a first medium.

In some embodiments, in the step of culturing the second TIL population in a second medium, the second medium further comprises anti-CD 3 and anti-CD 28 beads or antibodies.

In some embodiments, the anti-CD 3 and anti-CD 28 beads or antibodies comprise OKT-3 in a second medium.

According to some embodiments, the foregoing method further comprises cryopreserving the collected TIL population using a cryopreservation medium. In some embodiments, the cryopreservation medium is a dimethylsulfoxide-based cryopreservation medium. In other embodiments, the cryopreservation medium is CS10.

In some embodiments, the invention provides methods described in any of the preceding paragraphs above, modified where appropriate, such that the step of culturing the second TIL population in the second medium is performed for about 2 to 3 days.

In some embodiments, the invention provides methods described in any of the preceding paragraphs above, modified where appropriate, such that the step of culturing the second TIL population in the second medium is performed for about 3 to 4 days.

In some embodiments, the invention provides methods described in any of the preceding paragraphs above, modified where appropriate, such that the step of culturing the second TIL population in the second medium is performed for about 2 days.

In some embodiments, the invention provides methods described in any of the preceding paragraphs above, modified where appropriate, such that the step of culturing the second TIL population in the second medium is performed for about 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the second TIL population in the second medium is performed for about 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, such that the step of culturing the third or fourth TIL population in the second or third cell culture medium that applies occurs for about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days, as appropriate.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 9 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 10 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 11 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 12 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 13 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 14 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 9 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 10 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 11 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 12 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 13 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 9 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 9 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 9 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 9 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 10 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 10 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 10 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 11 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 11 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 12 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population in the second or third cell culture medium is performed for about 15 days.

According to some embodiments, any of the foregoing methods may be used to provide a population of autologous collected TILs for use in treating a human subject having cancer.

C. gene editing method

As described above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that are genetically modified (e.g., expression of immunomodulatory fusion proteins on their cell surfaces) by gene editing to enhance their therapeutic effects. Embodiments of the invention contemplate gene editing by nucleotide (RNA or DNA) insertion into a TIL population to promote expression of and inhibit expression of one or more proteins, and combinations thereof. Embodiments of the invention also provide methods of amplifying TIL into a therapeutic population, wherein the methods comprise gene editing of the TIL. There are several gene editing techniques available for genetically modifying a population of TILs, which are suitable for use in accordance with the present invention.

In some embodiments, the method of genetically modifying a population of TILs comprises the step of stably incorporating genes for producing more than one protein. In some embodiments, the method of genetically modifying a population of TILs comprises the step of retroviral transduction. In some embodiments, the method of genetically modifying a TIL population comprises the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine et al, proc. Natl. Acad. Sci. USA 2006,103,17372-77; zufferey et al, nature Biotechnology 1997,15,871-75; dull et al, J.Virol. 1998,72,8463-71 and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises the step of γ -retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, current protocols in molecular biology (Cur. Prot. Mol. Biol.) 1996,9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises the step of transposon mediated gene transfer. Transposon mediated gene transfer systems are known in the art and include those in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long term expression of the transposon does not occur in a transgenic cell, such as transposase provided as mRNA (e.g., mRNA comprising a cap and a poly a tail). Suitable transposon mediated gene transfer systems including salmon-type Tel-like transposases (SB or sleeping beauty transposases (e.g., SB10, SB11 and SB100 x)) and enzymes with increased engineered enzymatic activity are described, for example, in the following: hackett et al, molecular therapy 2010,18,674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

In some embodiments, the method of genetically modifying a population of TILs comprises the step of stably incorporating genes for producing or inhibiting (e.g., silencing) more than one protein. In some embodiments, the method of genetically modifying a population of TILs comprises the step of electroporation. Electroporation methods are known in the art and are described, for example, in the following: tsong journal of biophysics 1991,60,297-306 and U.S. patent application publication No. 2014/0227237A1, the respective disclosures of which are incorporated herein by reference. Other electroporation methods known in the art may be used, such as the electroporation methods described below: U.S. Pat. nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein a first pulse interval of two of the first set of at least three pulses is different from a second pulse interval of two of the second set of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, comprising the step of applying a series of at least three DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two, or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses such that the induced pores last for a relatively long period of time and maintain survival of the TIL. In some embodiments, the method of genetically modifying a TIL population comprises the step of calcium phosphate transfection. Methods of calcium phosphate transfection (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in the following: graham and van der Eb, virology 1973,52,456-467; wigler et al, 1979,76,1373-1376 in the national academy of sciences of the United states; chen and Okayarea, molecular cell biology 1987,7,2745-2752; and U.S. patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs comprises a step of lipofection. Liposome transfection methods, for example, using 1:1 (w/w) liposome formulations of the cationic lipids N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) in filtered water are known in the art and described in the following: rose et al, biotechnology 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA, 1987,84,7413-7417 and U.S. Pat. Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484 and 7,687,070, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises performing a transfection step using the method described in: U.S. patent nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of each of which are incorporated herein by reference.

According to some embodiments, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at more than one immune checkpoint gene. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on recognition of specific DNA sequences within the genome to target the nuclease domain to this location and mediate the creation of double-strand breaks at the target sequence. Double strand breaks in DNA then recruit endogenous repair systems (machinery) to the break site to mediate genome editing through non-homologous end joining (NHEJ) or Homology Directed Repair (HDR). Thus, repair of the break may result in the introduction of insertion/deletion mutations that disrupt (e.g., silence, inhibit or enhance) the target gene product.

The main classes of nucleases developed to enable site-specific genome editing include Zinc Finger Nucleases (ZFNs), transcription activator-like nucleases (TALENs) and CRISPR-associated nucleases (e.g., CRISPR/Cas 9). These nuclease systems can be broadly classified into two classes based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, while CRISPR systems (e.g., cas 9) target specific DNA sequences through short RNA guide molecules that base pair directly with the target DNA and through protein-DNA interactions. See, for example, cox et al, nature Medicine 2015, volume 21, phase 2.

Non-limiting examples of gene editing methods that may be used in accordance with the TIL amplification methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, embodiments of which are described in more detail below. According to some embodiments, the method for amplifying a TIL into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., process 2A) or as described in PCT/US2017/058610, PCT/US 2018/01605, or PCT/US2018/012633, wherein the method further comprises editing at least a portion of the TIL by one or more genes of the CRISPR method, the TALE method, or the ZFN method to produce a TIL that may provide enhanced therapeutic effects. According to some embodiments, the improved therapeutic effect of a genetically edited TIL may be assessed by comparing the genetically edited TIL to an unmodified TIL, e.g., by assessing in vitro effector function, cytokine profile, etc., as compared to an unmodified TIL.

In some embodiments of the invention, electroporation is used to deliver gene editing systems, such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow electroporation system. An example of a suitable flow electroporation system suitable for use in some embodiments of the invention is the commercially available MaxCyte STX system. There are several alternative commercially available electroporation devices that may be suitable for use in the present invention, such as AgilePulse systems available from BTX-Harvard Apparatus or ECM 830, cellaxess Elektra (Cellectricon), nuleofector (Lonza)/Amaxa), genePulser MXcell (bire (BIORAD), iPorator-96 (Primax) or siPORTer (Ambion) in some embodiments of the present invention, the electroporation system forms a closed sterile system with the remainder of the TIL amplification method.

In some embodiments, the gene editing system is delivered using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

D. transient cell modification

In some embodiments, the amplified TILs of the present invention are further manipulated to alter protein expression in a transient manner before, during, or after the amplification step, including during the closed aseptic manufacturing process (each as provided herein). In some embodiments, the invention includes transient cell modifications by nucleotide insertion (e.g., by ribonucleic acid (RNA) insertion, including insertion of messenger RNA (mRNA)) into the TIL population to promote expression or inhibit expression of more than one protein and simultaneously promote the combination of one set of proteins with inhibiting another set of proteins.

In some embodiments, the amplified TILs of the present invention undergo a transient change in protein expression. In some embodiments, a transient change in protein expression is made in the subject TIL population prior to the first amplification. In some embodiments, the transient change in protein expression is performed after the first amplification. In some embodiments, a transient change in protein expression is made in the subject TIL population prior to the second amplification. In some embodiments, the transient change in protein expression is performed after the second amplification.

In some embodiments, the transient change in protein expression causes transient expression of the immunomodulatory composition. In some embodiments, the immunomodulatory composition is an immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises a membrane anchor fused to an immunomodulatory agent. In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18 and IL-21. In some embodiments, the immunomodulator is an interleukin selected from the group consisting of: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is an interleukin selected from the group consisting of: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

As described herein, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been transiently modified by transient changes in protein expression to enhance their therapeutic effects. Embodiments of the invention encompass transient modifications by inserting nucleotides (e.g., RNA) into a TIL population for use in immunomodulating expression of a composition. Embodiments of the invention also provide methods of amplifying TIL into a therapeutic population, wherein the methods comprise transiently modifying TIL. There are several gene editing techniques available for transiently modifying a population of TILs, which are suitable for use in accordance with the present invention.

In some embodiments, the method of transiently altering protein expression in a population of TILs comprises contacting the TILs with a nucleic acid (e.g., mRNA) encoding an immunomodulatory composition, and then subjecting the cells to an electroporation step. Electroporation methods are known in the art and are described, for example, in the following: tsong journal of biophysics 1991,60,297-306 and U.S. patent application publication No. 2014/0227237A1, the respective disclosures of which are incorporated herein by reference. Other electroporation methods known in the art may be used, such as the electroporation methods described below: U.S. Pat. nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, including the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein a first pulse interval of two of the first set of at least three pulses is different from a second pulse interval of two of the second set of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, comprising the step of applying a series of at least three DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two, or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses such that the induced pores last for a relatively long period of time and maintain survival of the TIL.

In some embodiments, the method of transiently altering protein expression in a TIL population comprises the step of calcium phosphate transfection. Methods of calcium phosphate transfection (calcium phosphate nucleic acid precipitation, cell surface coating and endocytosis) are known in the art and are described in the following: graham and van der Eb, virology 1973,52,456-467; wigler et al, 1979,76,1373-1376 in the national academy of sciences of the United states; and Chen and Okayarea, molecular cell biology (mol. Cell. Biol.) 1987,7,2745-2752; and U.S. patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of transiently altering protein expression in a TIL population comprises a step of lipofection. Liposome transfection methods, for example, using 1:1 (w/w) liposome formulations of the cationic lipids N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) in filtered water are known in the art and are described in the following: rose et al, biotechnology 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA, 1987,84,7413-7417 and U.S. Pat. Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484 and 7,687,070, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of transiently altering protein expression in a TIL population comprises a transfection step using the method described in: U.S. patent nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of each of which are incorporated herein by reference. The TIL may be a first TIL population, a second TIL population, and/or a third TIL population as described herein.

In some embodiments, the transiently altered protein expression is performed using an SQZ carrier-free microfluidic platform. See, for example, international patent application publication No. WO 2013/059343A1, WO 2017/008063A1, or WO 2017/123663A1, or U.S. patent application publication No. US2014/0287509A1, US2018/0201889A1, or US2018/0245089A1, each of which is incorporated herein by reference in its entirety, especially with respect to the disclosure of microfluidic platforms for nucleic acid delivery. In the SQZ platform, delivery of nucleic acids encoding transiently expressed proteins is achieved by the micro-fluidic contraction to temporarily disrupt the cell membrane of the TIL for modification. The TIL may be a first TIL population, a second TIL population, and/or a third TIL population as described herein.

E. Immune checkpoint

According to a particular embodiment of the invention, the population of TILs is genetically edited to express one or more immunomodulatory compositions on the cell surface of the TIL cells in the population of TILs and to genetically modify one or more immune checkpoint genes in the population of TILs. In other words, in addition to modification of the population of TILs expressing one or more immunomodulatory compositions on the cell surface, DNA sequences encoding one or more immune checkpoints of the TILs within the TILs are permanently modified, e.g., inserted, deleted or substituted, in the genome of the TILs. An immune checkpoint is a molecule expressed by lymphocytes that modulates an immune response through an inhibitory or stimulatory pathway. In the case of cancer, the immune checkpoint pathway is typically activated to inhibit the anti-tumor response, i.e., certain immune checkpoints expressed by malignant cells inhibit anti-tumor immunity and favor cancer cell growth. See, for example, marin-Acevedo et al, journal of hematology and Oncology (Journal of Hematology & Oncology) (2018) 11:39. Thus, certain inhibitory checkpoint molecules serve as targets for the immunotherapy of the invention. According to particular embodiments, TIL is genetically engineered to block or stimulate certain immune checkpoint pathways, thereby enhancing the immune activity of the body against tumors.

As used herein, an immune checkpoint gene comprises a DNA sequence encoding an immune checkpoint molecule. According to particular embodiments of the invention, gene editing of the TIL during the TIL amplification method may result in silencing or reducing expression of more than one immune checkpoint gene in at least a portion of the therapeutic TIL population. For example, gene editing can result in silencing or reduction of expression of inhibitory receptors (e.g., PD-1 or CTLA-4) to enhance immune responses.

The most widely studied checkpoints include programmed cell death receptor-1 (PD-1) and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), which are inhibitory receptors on immune cells that inhibit important effector functions (e.g., activation, proliferation, cytokine release, cytotoxicity, etc.) upon interaction with inhibitory ligands. In addition to PD-1 and CTLA-4, many checkpoint molecules have become potential targets for immunotherapy, as discussed in more detail below.

Non-limiting examples of immune checkpoint genes that can be silenced or inhibited by permanent gene editing of the TIL of the present invention include PD-1、CTLA-4、LAG-3、HAVCR2(TIM-3)、Cish、TGFβ、PKA、CBL-B、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、BTLA、CD160、TIGIT、TET2、BAFF(BR3)、CD96、CRTAM、LAIR1、SIGLEC7、SIGLEC9、CD244、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT1、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2、GUCY1B3、TOX、SOCS1、ANKRD11 and BCOR. For example, immune checkpoint genes that may be silenced or inhibited in the TIL of the present invention may be selected from: PD-1, CTLA-4, LAG-3, TIM-3, cish, CBL-B, TIGIT, TET2, TGF beta and PKA. BAFF (BR 3) is described in published Bloom et al, journal of immunotherapy (j.immunother.), 2018. According to another example, an immune checkpoint gene that may be silenced or inhibited in the TIL of the present invention may be selected from: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGF beta R2, PRA, CBLB, BAFF (BR 3), and combinations thereof.

(b) Adding tumor fragments into the closed system;

(e) Performing aseptic electroporation on the second population of TILs to effect transfer of the at least one gene editor into the plurality of cells in the second population of TILs;

(f) Standing the second TIL population for about 1 day;

(g) Performing a second amplification by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and Antigen Presenting Cells (APC), producing a third TIL population, wherein the second amplification is performed for about 7 to 11 days, the second amplification is performed in a closed container providing a second gas permeable surface area, and the transition from step (f) to step (g) is performed without opening the system;

(h) Collecting the third TIL population obtained from step (g) to obtain a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, the collected TIL population being a therapeutic TIL population;

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

(J) Optionally, cryopreserving the collected TIL population using a cryopreservation medium,

Wherein the electroporation step comprises delivering at least one gene-editor system selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system effects expression of at least one immunomodulatory composition on a cell surface of a plurality of cells in a second TIL population, inhibiting expression of a molecule selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGF beta R2, PRA, CBLB, BAFF (BR 3), and combinations thereof. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

1.PD-1

One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD 1 or PD-1, also known as PDCD 1), a member of the CD28 superfamily of T cell modulators. Its ligands PD-L1 and PD-L2 are expressed on various tumor cells, including melanoma. The interaction of PD-1 with PD-L1 can inhibit T cell effector function, leading to T cell depletion in a chronically stimulated environment and induction of T cell apoptosis in a tumor microenvironment. PD1 may also play a role in tumor-specific escape immune surveillance.

According to particular embodiments, the compositions and methods of the invention silence or reduce the expression of PD1 in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be performed according to any embodiment of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises gene editing at least a portion of the TILs by silencing or inhibiting expression of PD 1. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., PD 1). For example, the CRISPR method, TALE method, or zinc finger method can be used to silence or reduce expression of PD1 in TIL.

2.CTLA-4

CTLA-4 expression on activated T cells is induced upon T cell activation and competes for binding with antigen CD80 and CD86 activated by antigen presenting cells. The interaction of CTLA-4 with CD80 or CD86 may cause T cell inhibition and serve to maintain the balance of immune responses. However, inhibiting CTLA-4 interaction with CD80 or CD86 can prolong T cell activation and thus increase the level of immune response against cancer antigens.

According to particular embodiments, the compositions and methods of the invention silence or reduce expression of CTLA-4 in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a population of therapeutic TILs can be performed according to any of the embodiments of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of CTLA-4 in the TILs. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., CTLA-4). For example, the CRISPR method, TALE method, or zinc finger method can be used to silence or inhibit CTLA-4 expression in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

3.LAG-3

After class II histocompatibility complex (MHC) engagement, lymphocyte activator gene-3 (LAG-3, cd 223) is expressed by T cells and Natural Killer (NK) cells. Although the mechanism is not yet defined, its modulation may cause negative regulation of T cell function, preventing tissue damage and autoimmunity. LAG-3 and PD-1 are commonly co-expressed and up-regulated on TIL, leading to immune depletion and tumor growth. Thus, LAG-3 blockade may improve anti-tumor responses. See, for example, marin-Acevedo et al, J.Hematology and oncology (2018) 11:39.

According to particular embodiments, the compositions and methods of the present invention silence or reduce the expression of LAG-3 in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any embodiment of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or fig. 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of LAG-3 in the TILs. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., LAG-3). According to particular embodiments, the CRISPR method, TALE method, or zinc finger method may be used to silence or inhibit LAG-3 expression in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

4.TIM-3

T cell immunoglobulin-3 (TIM-3) is a direct negative regulator of T cells and is expressed on NK cells and macrophages. TIM-3 indirectly promotes immunosuppression by inducing expansion of myeloid-derived suppressor cells (MDSCs). It was found that its content on dysfunctional and depleted T cells was specifically elevated, indicating an important role in malignancy.

According to particular embodiments, the compositions and methods of the present invention silence or reduce the expression of TIM-3 in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be performed according to any of the embodiments of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or fig. 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of TIM-3 in the TILs. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., TIM-3). For example, the CRISPR method, TALE method, or zinc finger method can be used to silence or inhibit expression of TIM-3 in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

5.Cish

Cish members of the cytokine signaling suppressor (SOCS) family, are induced by TCR stimulation in cd8+ T cells and inhibit their functional avidity for tumors. The gene deletion of Cish in cd8+ T cells can enhance its expansion, functional avidity, and cytokine versatility, leading to significant and durable regression of existing tumors. See, e.g., palmer et al, journal of experimental medicine (Journal of Experimental Medicine), 212 (12): 2095 (2015).

According to particular embodiments, the compositions and methods of the present invention silence or reduce expression of Cish in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any embodiment of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of Cish in the TILs. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., cish). For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit expression of Cish in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

6.TGFβ

Tgfβ signaling pathways have a variety of functions in regulating cell growth, differentiation, apoptosis, motility and invasion, extracellular matrix production, angiogenesis and immune responses. Deregulation of tgfβ signalling is common in tumours and has an important role in tumorigenesis, development and metastasis. At the microenvironment level, the tgfβ pathway contributes to the creation of a microenvironment conducive to tumor growth and metastasis in all carcinogenesis. See, e.g., neuzillet et al, pharmacology & Therapeutics, volume 147, pages 22-31 (2015).

According to particular embodiments, the compositions and methods of the invention silence or reduce expression of tgfβ in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any of the embodiments of the methods described herein (e.g., the methods shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of tgfβ in the TILs. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., tgfβ). For example, the CRISPR method, TALE method, or zinc finger method can be used to silence or inhibit expression of tgfβ in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

In some embodiments, tgfβr2 (tgfβreceptor 2) can be inhibited by silencing tgfβr2 using a CRISPR/Cas9 system or by using a tgfβr2 dominant negative extracellular trap (trap) using methods known in the art.

7.PKA

Protein Kinase A (PKA) is a well known member of the serine-threonine protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multi-unit protein kinase that mediates G-protein coupled receptor signaling through its activation upon cAMP binding. It involves the control of a variety of cellular processes from metabolism to ion channel activation, cell growth and differentiation, gene expression and apoptosis. Importantly, PKA is associated with the development and progression of many tumors. See, e.g., sapio et al, journal of experimental and clinical science (EXCLI Journal); 2014;13:843-855.

According to particular embodiments, the compositions and methods of the invention silence or reduce the expression of PKA in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any embodiment of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of PKA in the TILs. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., PKA). For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit PKA expression in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

8.CBLB

CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and is a negative regulator of T cell activation. Bachmaier et al, nature, 2000,403,211-216; wallner et al, clinical and developing immunology (clin. Dev. Immunol.) 2012,692639.

According to particular embodiments, the compositions and methods of the present invention silence or reduce the expression of CBLB in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any embodiment of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or fig. 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of CBLB in the TILs. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., CBLB). For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit PKA expression in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, a TALEN gene knockout is used to silence CBLB. In some embodiments, the CBLB is silenced using TALE-KRAB transcription inhibitor gene knock-in. Further details on these methods can be found in Boettcher and McManus, molecular cell Review, 2015,58,575.

9.TIGIT

T cell immunoreceptors with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains or TIGIT are transmembrane glycoprotein receptors with an Ig-like V-type domain and ITIM in their cytoplasmic domains. Khalil et al, progress of cancer research (ADVANCES IN CANCER RESEARCH), 2015,128,1-68; yu et al, (Nature Immunology) Nature immunology, 2009, volume 10, no. 1, 48-57.TIGIT is expressed by some T cells and natural killer cells. Furthermore, TIGIT has been shown to be overexpressed on antigen-specific cd8+ T cells and cd8+ TIL (especially from subjects with melanoma). Studies have shown that TIGIT pathways contribute to tumor immune evasion and that TIGIT inhibition can increase T cell activation and proliferation in response to polyclonal and antigen specific stimuli. Khalil et al, progress of cancer research, 2015,128,1-68. Furthermore, co-blocking TIGIT with PD-1 or TIM3 showed a synergistic effect against solid tumors in the mouse model. As above; see also Kurtulus et al, journal of clinical research (The Journal of Clinical Investigation), 2015, volume 125, 11, 4053-4062.

According to particular embodiments, the compositions and methods of the present invention silence or reduce the expression of TIGIT in the TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any of the embodiments of the methods described herein (e.g., the methods shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of TIGIT in the TILs. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., TIGIT). For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit TIGIT expression in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

10.TOX

Thymocytes were selected from the relevant high mobility group (high mobility group) (HMG) box (TOX) as transcription factors comprising HMG box DNA binding domains. TOX is a member of the HMG box superfamily, which is thought to bind DNA in a sequence-independent, but structure-dependent manner.

TOX has been identified as an important regulator of tumor-specific CD8 ⁺ T cell dysfunction or T cell depletion and found to arrange CD8 ⁺ T cell depletion in a transcriptional and epigenetic manner, as described, for example, in Scott et al, nature, 2019,571,270-274 and Khan et al, nature, 2019,571,211-218, each of which is incorporated herein by reference in its entirety. TOX has also been found to be an important factor in the development of T cell dysfunction and maintenance of T cell depletion during chronic infections, as described in Alfei et al, nature, 2019,571,265-269, which is incorporated herein by reference in its entirety. TOX is abundantly expressed in dysfunctional or depleted T cells from tumor and chronic viral infections. Ectopic expression of TOX in effector T cells in vitro induces transcriptional programs associated with T cell depletion, whereas deletion of TOX in T cells abrogates T depletion programs.

According to certain embodiments, the compositions and methods of the invention silence or reduce expression of TOX in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any embodiment of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or fig. 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on a cell surface and silencing or inhibiting expression of TOX. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at immune checkpoint genes (e.g., TOX). For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit expression of TOX in TIL. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

F. Overexpression of costimulatory receptors or adhesion molecules

According to other embodiments, gene editing of the TIL during the TIL amplification method may cause expression of at least one immunomodulatory composition at the cell surface, resulting in enhanced expression of one or more co-stimulatory receptors, adhesion molecules, and/or cytokines in at least a portion of the therapeutic TIL population. For example, gene editing may result in enhanced expression of a co-stimulatory receptor, adhesion molecule or cytokine, i.e., over-expression compared to the expression of a co-stimulatory receptor, adhesion molecule or cytokine that has not been genetically modified. Non-limiting examples of co-stimulatory receptors, adhesion molecules or cytokine genes that may exhibit enhanced expression by the permanent gene editing TILs of the present invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1.

1.CCR

In order for adoptive T cell immunotherapy to be effective, appropriate migration of T cells into the tumor by chemokines is required. The matching between chemokines secreted by tumor cells, chemokines present in the periphery and chemokine receptors expressed by T cells is important for the successful migration of T cells into the tumor bed.

According to particular embodiments, the gene editing methods of the invention may be used to increase expression of certain chemokine receptors (e.g., more than one of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR 1) in TIL. Overexpression of CCR can help promote effector function and proliferation of TIL after adoptive transfer.

According to particular embodiments, the compositions and methods of the invention enhance expression of more than one of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1 in TIL. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs can be performed according to any of the embodiments of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and to enhance expression of more than one of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1 in the TILs. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at the chemokine receptor gene. For example, the CRISPR method, TALE method, or zinc finger method can be used to enhance expression of certain chemokine receptors in TIL.

In some embodiments, CCR4 and/or CCR5 adhesion molecules are inserted into the TIL population using a gamma-retrovirus or lentiviral method as described herein. In some embodiments, the CXCR2 adhesion molecule is inserted into the TIL population using the gamma-retrovirus or lentivirus method described below: forget et al, leading edge immunology (Frontiers Immunology), 2017,8,908 or Peng et al, clinical cancer research (clin. Cancer res.) 2010,16,5458, the disclosures of which are incorporated herein by reference.

(b) Adding tumor fragments into the closed system;

(d) Stimulating the second population of TILs by adding OKT-3 and incubating for about 1 to 3 days to obtain a second population of TILs, the transitioning from step (c) to step (d) being performed without opening the system;

(f) Standing the second TIL population for about 1 day;

(h) Collecting the third TIL population obtained from step (g) to obtain a collected TIL population, wherein the transition from step (g) to step (h) occurs without turning on the system, the collected TIL population being a therapeutic TIL population;

Wherein the electroporation step comprises delivering at least one gene-editor system selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene-editor system effects expression of at least one immunomodulatory composition on a cell surface of a plurality of cells in a second TIL population, inhibits expression of PD-1 and optionally LAG-3 present, and further wherein the at least one gene-editor system effects expression of CXCR2 adhesion molecules at a cell surface of a plurality of cells in a second TIL population or inserts CXCR2 adhesion molecules into a first TIL population, a second TIL population, or a collected TIL population by a gamma retrovirus or lentiviral method. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein the electroporation step comprises delivering at least one gene-editor system selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system effects expression of at least one immunomodulatory composition on a cell surface of a plurality of cells in a second TIL population, inhibits expression of PD-1 and optionally LAG-3 present, and further wherein the at least one gene editor system effects expression of CCR4 and/or CCR5 adhesion molecules at a cell surface of a plurality of cells in a second TIL population or inserts CXCR2 adhesion molecules into a first TIL population, a second TIL population, or a collected TIL population by a gamma retrovirus or lentiviral method. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

(b) Adding tumor fragments into the closed system;

(d) Stimulating the second population of TILs by adding OKT-3 and incubating for about 1 to 3 days to obtain a second population of TILs, wherein the transitioning from step (c) to step (d) occurs without opening the system;

(f) Standing the second TIL population for about 1 day;

(g) Performing a second amplification by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and Antigen Presenting Cells (APC), producing a third TIL population, wherein the second amplification is performed for about 7 to 11 days, wherein the second amplification is performed in a closed container providing a second gas permeable surface area, the transition from step (f) to step (g) being performed without opening the system;

Wherein the electroporation step comprises delivering at least one gene-editor system selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system effects expression of at least one immunomodulatory composition on a cell surface of a plurality of cells in a second TIL population, inhibits expression of PD-1 and optionally LAG-3 present, and further wherein the at least one gene editor system effects expression of an adhesion molecule selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof at a cell surface of a plurality of cells in a second TIL population or inserts the adhesion molecule into the first TIL population, the second TIL population, or the collected TIL population by a gamma retrovirus or lentiviral method. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

2. Interleukin

According to other embodiments, the gene editing methods of the invention can be used to increase the expression of certain interleukins (e.g., more than one of IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, and IL-21). Certain interleukins have been shown to enhance T cell effector function and mediate tumor control.

According to particular embodiments, the compositions and methods of the present invention enhance the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, and IL-21 in TIL. For example, a method of amplifying tumor-infiltrating lymphocytes (TILs) into a population of therapeutic TILs can be performed according to any embodiment of the methods described herein (e.g., the method shown in process 2A, process Gen 3, or figures 34 and 35), wherein the method comprises genetically editing at least a portion of the TILs by enhancing expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, and IL-21. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-or single-stranded breaks at the interleukin gene. For example, the CRISPR method, TALE method, or zinc finger method can be used to enhance the expression of certain interleukins in TIL.

(b) Adding tumor fragments into the closed system;

(e) Performing aseptic electroporation on the second population of TILs to effect transfer of the at least one gene editor;

(f) Leaving the second TIL population to stand for about 1 day to become a plurality of cells in the second TIL population;

(h) Collecting the therapeutic TIL population obtained from step (g) to obtain a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, the collected TIL population being a therapeutic TIL population;

Wherein the electroporation step comprises delivering at least one gene-editor system selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system effects expression of at least one immunomodulatory composition on a cell surface of a plurality of cells in a second TIL population, inhibits expression of PD-1 and optionally LAG-3 present, and further wherein the at least one gene editor system effects expression of an interleukin selected from IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, and combinations thereof at a cell surface of a plurality of cells in a second TIL population or inserts an interleukin into a first TIL population, a second TIL population, or a collected TIL population by a gamma retrovirus or lentiviral method. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the cytokine is selected from: IL-12, IL-15, IL-18 and IL-21.

3. Gene editing method

As described above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that are genetically modified by gene editing to enhance their therapeutic effects, including TILs that are modified by transient gene editing to transiently alter protein expression in the modified TILs. Embodiments of the invention contemplate gene editing by nucleotide (RNA or DNA) insertion into a TIL population to promote expression of and inhibit expression of more than one protein, and combinations thereof. Embodiments of the invention also provide methods for amplifying TILs into a therapeutic population, wherein the methods comprise gene editing the TILs, or wherein the methods comprise transiently gene editing the TILs to transiently alter the expression of proteins in the modified TILs. There are several gene editing techniques available for genetically modifying a population of TILs, including transient gene editing techniques for transiently altering the expression of proteins in a population of TILs, which are suitable for use in accordance with the present invention.

In some embodiments, the method of genetically modifying a population of TILs comprises the step of stably incorporating genes for producing more than one protein. In some embodiments, the method of genetically modifying a population of TILs comprises the step of retroviral transduction. In some embodiments, the method of genetically modifying a TIL population comprises the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in the following: levine et al, 2006,103,17372-77, proc. Natl. Acad. Sci. USA; zufferey et al, nature Biotechnology (Nat. Biotechnol.) 1997,15,871-75; dull et al J.virology 1998,72,8463-71 and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises the step of γ -retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, current protocols in molecular biology, 1996,9.9.1-9.9.16, the disclosures of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises the step of transposon mediated gene transfer. Transposon mediated gene transfer systems are known in the art and include those in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long term expression of the transposase does not occur in transgenic cells, such as transposase provided as mRNA (e.g., mRNA comprising a cap and a poly a tail). Suitable transposon mediated gene transfer systems including salmon-type Tel-like transposases (SB or sleeping beauty transposases (e.g., SB10, SB11 and SB100 x)) and enzymes with increased engineered enzymatic activity are described, for example, in the following: hackett et al, molecular therapy 2010,18,674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

In some embodiments, the method of genetically modifying a population of TILs comprises the step of stably incorporating genes for producing or inhibiting (e.g., silencing) more than one protein. In some embodiments, the method of genetically modifying the TIL population (e.g., the method of transiently modifying by transiently altering protein expression) comprises an electroporation step. Electroporation methods are known in the art and are described, for example, in the following: tsong journal of biophysics 1991,60,297-306 and U.S. patent application publication No. 2014/0227237A1, the respective disclosures of which are incorporated herein by reference. Other electroporation methods known in the art may be used, such as the electroporation methods described below: U.S. Pat. nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein a first pulse interval of two of the first set of at least three pulses is different from a second pulse interval of two of the second set of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, comprising the step of applying a series of at least three DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two, or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses such that the induced pores last for a relatively long period of time and maintain survival of the TIL.

In some embodiments, the method of genetically modifying the TIL population (e.g., the method of transiently modifying by transiently altering protein expression) comprises a calcium phosphate transfection step. Methods of calcium phosphate transfection (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in the following: graham and van der Eb, virology 1973,52,456-467; wigler et al, 1979,76,1373-1376 in the national academy of sciences of the United states; chen and Okayarea, molecular cell biology 1987,7,2745-2752; and U.S. patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying the TIL population (e.g., the method of transiently modifying by transiently altering protein expression) comprises a liposome transfection step. Liposome transfection methods, for example, using 1:1 (w/w) liposome formulations of the cationic lipids N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) in filtered water are known in the art and are described in the following: rose et al, biotechnology 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA, 1987,84,7413-7417 and U.S. Pat. Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484 and 7,687,070, the disclosures of each of which are incorporated herein by reference. In some embodiments, methods of genetically modifying a population of TILs (e.g., methods of transiently genetically modifying by transiently altering protein expression) include the use of U.S. patent No. 5,766,902; 6,025,337 th sheet; 6,410,517 th sheet; 6,475,994 th sheet; and the transfection steps of the methods described in 7,189,705, the respective disclosures of which are incorporated herein by reference.

According to some embodiments, the gene editing process may include the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at more than one immune checkpoint gene. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on recognition of specific DNA sequences within the genome to target the nuclease domain to this location and mediate the creation of double-strand breaks at the target sequence. Double strand breaks in DNA then recruit endogenous repair mechanisms to the break site to mediate genome editing through non-homologous end joining (NHEJ) or Homology Directed Repair (HDR). Thus, repair of the break may result in the introduction of insertion/deletion mutations that disrupt (e.g., silence, inhibit or enhance) the target gene product.

The main classes of nucleases developed to enable site-specific genome editing include Zinc Finger Nucleases (ZFNs), transcription activator-like nucleases (TALENs) and CRISPR-associated nucleases (e.g., CRISPR/Cas 9). These nuclease systems can be broadly classified into two classes based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, whereas CRISPR systems (e.g., cas 9) target specific DNA sequences through short RNA guide molecules that base pair directly with target DNA and through protein-DNA interactions. See, for example, cox et al, nature Medicine 2015, volume 21, phase 2.

Non-limiting examples of gene editing methods that may be used in accordance with the TIL amplification methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, embodiments of which are described in more detail below. According to some embodiments, the method of amplifying a TIL into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., process 2A) or as described in PCT/US2017/058610, PCT/US 2018/01605, or PCT/US2018/012633, wherein the method further comprises editing at least a portion of the TIL by one or more genes of the CRISPR method, TALE method, or ZFN method to produce a TIL that may provide enhanced therapeutic effects. According to some embodiments, the improved therapeutic effect of a genetically edited TIL may be assessed by comparing the genetically edited TIL to an unmodified TIL, e.g., by assessing in vitro effector function, cytokine profile, etc., as compared to an unmodified TIL.

CRISPR method

The method for amplifying a TIL into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., process 2A) or as described in PCT/US2017/058610, PCT/US 2018/01605, or PCT/US2018/012633, wherein the method further comprises editing at least a portion of the TIL by a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf 1) gene. According to particular embodiments, the use of a CRISPR method during a TIL amplification procedure may cause expression of one or more immunomodulatory compositions at the cell surface of at least a portion of a therapeutic TIL population, optionally causing silencing or reduction of one or more immune checkpoint genes. Or the use of a CRISPR method during a TIL amplification procedure may cause expression of at least one immunomodulatory composition at the cell surface of at least a portion of a therapeutic TIL population, optionally causing enhancement of more than one immune checkpoint gene. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

CRISPR stands for "clustered regularly interspaced short palindromic repeats". The method of gene editing using a CRISPR system is also referred to herein as the CRISPR method. CRISPR systems can be divided into two main categories, namely category 1 and category 2, which are further divided into different types and subtypes. Classification of CRISPR systems is based on effector Cas proteins capable of cleaving specific nucleic acids. In a class 1 CRISPR system, the effector moiety consists of a polyprotein complex, whereas a class 2 system uses only one effector protein. Class 1 CRISPR includes types I, III and IV, and class 2 CRISPR includes types II, V and VI. Although any of these types of CRISPR systems may be used according to the present invention, the preferred three types of CRISPR systems used according to the present invention include RNA and Cas proteins: type I (exemplified by Cas 3), type II (exemplified by Cas 9), and type III (exemplified by Cas 10). Type II CRISPR is one of the most well characterized systems.

CRISPR technology adapts the natural defense mechanisms from bacteria and archaea (domains of unicellular microorganisms). These organisms use CRISPR-derived RNAs and various Cas proteins (including Cas 9) to prevent attack by viruses and other exosomes by chopping and disrupting the DNA of the foreign intruder. CRISPR is a DNA-specific region with two unique features: nucleotide repeats and spacers are present. The repeated sequence of nucleotides is distributed throughout the CRISPR region with short foreign DNA segments (spacers) interspersed in the repeated sequence. In a type II CRISPR/Cas system, a spacer is integrated within the CRISPR genomic locus and transcribed and processed into a short CRISPR RNA (crRNA). These crrnas anneal to transactivation crRNA (tracrRNA), which directs sequence-specific cleavage of Cas proteins and silencing of pathogenic DNA. Target recognition by Cas9 proteins requires a "seed" sequence within the crRNA and a conserved Protospacer Adjacent Motif (PAM) sequence containing dinucleotides upstream of the crRNA binding region, so the CRISPR/Cas system can be re-targeted by redesigning the crRNA to cleave almost any DNA sequence. Thus, according to certain embodiments, cas9 acts as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA recognition. Crrnas and tracrrnas in the original system can be reduced to about 100 nucleotide single guide RNAs (sgrnas) for genetic engineering. sgrnas are synthetic RNAs that include the necessary scaffold sequences for Cas binding and a user-defined about 17-to 20-nucleotide spacer that defines the genomic target to be modified. Thus, a user can alter the genomic target of the Cas protein by altering the sequence of interest present in the sgRNA. The CRISPR/Cas system can be carried directly by human cells by co-delivering plasmids expressing Cas9 endonuclease and RNA components (e.g., sgrnas). Different Cas protein variants may be used to reduce targeting limitations (e.g., heterologous homologs of Cas9, such as Cpf 1).

According to some embodiments, an engineered, programmable, non-naturally occurring type II CRISPR-Cas system comprises a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a TIL, wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule, the Cas9 protein cleaves the DNA molecule thereby altering expression of the at least one immune checkpoint molecule; the Cas9 protein and the guide RNA do not naturally co-exist. According to some embodiments, the expression of two or more immune checkpoint molecules is altered. According to some embodiments, the guide RNA comprises a guide sequence fused to a tracr sequence. For example, the guide RNA may comprise crRNA-tracrRNA or sgRNA. According to aspects of the invention, the terms "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence comprising a guide sequence, which is a sequence of about 17-20bp within the guide RNA that specifies the target site.

According to embodiments of the invention, cas9 variants with improved target specificity compared to Cas9 may also be used. Such variants may be referred to as high fidelity Cas-9. According to some embodiments, a double nicking enzyme method may be utilized, wherein two nicking enzymes targeting the reverse DNA strand produce DSBs within the target DNA (commonly referred to as double nicking or double nicking enzyme CRISPR systems). For example, this method can involve a mutation in one of the two Cas9 nuclease domains, transitioning Cas9 from nuclease to nickase. Non-limiting examples of high-fidelity Cas9 include eSpCas, spCas9-HF1, and HypaCas. Such variants may reduce or eliminate undesirable changes at non-target DNA sites. See, for example, SLAYMAKER IM et al, science, 2015, 12, 1, KLEINSTIVER BP et al, nature, 2016, 1, 6, and Ran et al, nature laboratory Manual (Nat Protoc.), 2013, 11; 8 (11): 2281-2308, the disclosure of which is incorporated herein by reference.

Furthermore, according to particular embodiments, cas9 scaffolds that improve gene delivery to cells and improve target specificity in cells, such as the scaffolds disclosed in U.S. patent application publication 2016/0102324, which is incorporated herein by reference, may be used. For example, the Cas9 scaffold may include up>A RuvC motif as defined by (D- [ I/L ] -G-X-S-X-G-W-up>A) and/or up>A HNH motif as defined by (Y-X-D-H-X-P-X-S-X-D-X-S), wherein X represents any of 20 naturally occurring amino acids and [ I/L ] represents isoleucine or leucine. The HNH domain is responsible for cleaving one strand of the target dsDNA and the RuvC domain involves cleavage of the other strand of the dsDNA. Thus, each of these domains cleaves one strand of target DNA within the immediately preceding spacer (protospacer) of PAM, causing blunt-ended cleavage (blunt cleaning) of the DNA. These motifs can be combined with each other to create a more compact and/or more specific Cas9 scaffold. Furthermore, motifs can be used to generate split (split) Cas9 proteins (i.e., reduced or truncated versions of Cas9 proteins or Cas9 variants comprising RuvC domains or HNH domains) that are separated into two separate RuvC and HNH domains, which can collectively or individually process target DNA.

According to particular embodiments, the CRISPR method comprises silencing or reducing expression of one or more immune checkpoint genes in the TIL by introducing a Cas9 nuclease and a guide RNA (e.g., crRNA-tracrRNA or sgRNA) containing a sequence of about 17 to 20 nucleotides specific for a target DNA sequence of an immune checkpoint gene. The guide RNA may be delivered in RNA form or by transformation of a plasmid with the coding sequence of the guide RNA under a promoter. Based on the target sequence defined by the sgrnas, the CRISPR/Cas enzyme introduces Double Strand Breaks (DSBs) at specific locations. DSBs in cells can be repaired by non-homologous end joining (NHEJ, a mechanism that typically causes insertions or deletions (indels) in DNA). Insertions/deletions often cause frame shifts, producing a loss-of-function allele; for example, by causing premature stop codons in the Open Reading Frame (ORF) of the target gene. According to certain embodiments, the result is a loss-of-function mutation within the target immune checkpoint gene.

Alternatively, instead of NHEJ, DSBs induced by CRISPR/Cas enzymes can be repaired by Homology Directed Repair (HDR). Although NHEJ-mediated SB repair typically breaks the open reading frame of a gene, homology Directed Repair (HDR) can be used to produce specific nucleotide changes ranging from single nucleotide changes to large insertions. According to some embodiments, the immune checkpoint genes are genetically edited using HDR by delivering a DNA repair template containing the desired sequence into the TIL with sgRNA and Cas9 or Cas9 nickase. The repair template preferably contains the desired editing and other homologous sequences immediately upstream and downstream of the target gene (commonly referred to as left and right homology arms).

According to particular embodiments, an enzymatically inactive version of Cas9 (readcas 9 or dCas 9) can be targeted to the transcription start site to inhibit transcription by blocking initiation. Thus, the target immune checkpoint genes can be inhibited without using DSBs. dCas9 molecules retain the ability to bind target DNA based on sgRNA targeting sequences. According to some embodiments of the invention, the CRISPR method comprises silencing or reducing expression of one or more immune checkpoint genes by inhibiting or preventing transcription of a target gene. For example, a CRISPR method can include fusing a transcriptional repressor domain (e.g., a Kruppel-related cassette (KRAB) domain) with an enzymatically inactive version of Cas9, thereby forming, e.g., dCas9-KRAB, which targets the transcription start site of an immune checkpoint gene, causing repression or prevention of transcription of the gene. Preferably, the inhibitor domain is targeted to a window downstream (e.g., about 500bp downstream) of the transcription initiation site. This approach, which may be referred to as CRISPR interference (CRISPRi), causes stable gene knockdown by reduced transcription of the target RNA.

According to particular embodiments, an enzymatically inactive version of Cas9 (readcas 9 or dCas 9) may be targeted to the transcription start site to activate transcription. This method may be referred to as CRISPR activation (CRISPRa). According to some embodiments, the CRISPR method increases the expression of more than one immune checkpoint gene by activating transcription of the target gene. According to such embodiments, the target immune checkpoint genes may be activated without the use of DSBs. CRISPR methods can include targeting a transcriptional activation domain to a transcriptional start site; for example, activation of gene transcription is caused by fusion of a transcriptional activator (e.g., VP 64) with dCS 9, thereby forming, e.g., dCS 9-VP64, which targets the transcriptional start site of the immune checkpoint gene. Preferably, the activator subdomain is targeted to a window at about 50 to 400bp upstream, e.g., downstream, of the transcription initiation site.

Other embodiments of the invention may utilize activation strategies developed for the potent activation of target genes in mammalian cells. Non-limiting examples include co-expression of dCas9 fused to epitope-tagged dCas9 and antibody-activator effector proteins (e.g., sunTag systems), to multiple different tandem activation domains (e.g., dCas 9-VPR), or dCas9-VP64 with co-expression of modified scaffold gRNA and other RNA-binding co-activators (e.g., SAM activators).

According to other embodiments, CRISPR-mediated genome editing methods known as CRISPR-assisted rational protein engineering (CARPE) may be used in accordance with embodiments of the present invention, as disclosed in U.S. patent No. 9,982,278, which is incorporated herein by reference. CARPE relates to the generation of "donor" and "target" libraries that incorporate directed mutations from single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) editing cassettes directly into the genome. Construction of the donor pool involves co-transformation of rationally designed editing oligonucleotides and guide RNAs (grnas) that hybridize to target DNA sequences into cells. The edited oligonucleotide is designed to couple the deletion or mutation of PAM with mutations at more than one desired codon in adjacent genes. This enables the generation of an entire donor pool in a single transformation. The donor pool is retrieved by amplification of the recombinant chromosome (e.g. by PCR reaction) using the synthetic features from the edited oligonucleotide, i.e. the second PAM deletion or mutation incorporated simultaneously at the 3' end of the gene. This covalently couples the codon target mutations for PAM deletions. The donor pool is then co-transformed with the target gRNA vector into cells to produce a population of cells expressing a rationally designed protein pool.

According to other embodiments, a method for trackable, precise genome editing using a CRISPR-mediated system, known as genome engineering by trackable CRISPR-rich recombination engineering (GEn-TraCER), as disclosed in U.S. patent No. 9,982,278, which is incorporated herein by reference, may be used in accordance with embodiments of the present invention. The GEn-TraCER method and vector combines an editing cassette with a gene encoding a gRNA on a single vector. The cassette contains the desired mutation and PAM mutation. A vector that can also encode Cas9 is introduced into a cell or cell population. Expression of the CRISPR system in this activated cell or cell population causes the gRNA to recruit Cas9 to the target region where dsDNA fragmentation occurs, enabling integration of the PAM mutation.

Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TIL by CRISPR methods include PD-1、CTLA-4、LAG-3、HAVCR2(TIM-3)、Cish、TGFβ、PKA、CBL-B、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、BTLA、CD160、TIGIT、TET2、CD96、CRTAM、LAIR1、SIGLEC7、SIGLEC9、CD244、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT1、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2、GUCY1B3、TOX、SOCS1、ANKRD11 and BCOR.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TIL by CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering expression of target gene sequences by CRISPR methods and which can be used in accordance with embodiments of the present invention are described in U.S. Pat. nos. 8,697,359; 8,993,233 th sheet; 8,795,965 th sheet; 8,771,945 th sheet; 8,889,356 th sheet; 8,865,406 th sheet; 8,999,641 th sheet; 8,945,839 th sheet; 8,932,814 th sheet; 8,871,445 th sheet; 8,906,616 th sheet; and 8,895,308, incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are available from companies such as gold (GenScript).

In some embodiments, genetic modification of a TIL population as described herein may be performed using a CRISPR/Cpf1 system as described in U.S. patent No. 9,790,490, the disclosure of which is incorporated herein by reference. The CRISPR/Cpf1 system differs functionally from the CRISPR-Cas9 system in that Cpf 1-related CRISPR arrays (array) can be processed to mature crrnas without additional tracrRNA. Crrnas used in CRISPR/Cpf1 systems have a spacer or guide sequence and a direct repeat sequence. The Cpf1p-crRNA complex formed using this method is sufficient in itself to cleave the target DNA.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

(h) Collecting the therapeutic TIL population obtained from step (g) to obtain a collected TIL population, wherein the transition from step (g) to step (h) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population;

Wherein the electroporation step comprises delivering at least one gene-editor system selected from the group consisting of: clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 9 systems and CRISPR/Cpf1 systems, wherein the at least one gene editor system effects expression of at least one immunomodulatory composition on a cell surface of a plurality of cells in a second TIL population, modulating expression of at least one checkpoint protein.

(b) Adding tumor fragments into the closed system;

(d) Stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days to obtain a second population of TILs, wherein the transition from step (c) to step (d) is performed without opening the system;

(f) Standing the second TIL population for about 1 day;

Wherein the electroporation step comprises delivering at least one gene-editor system selected from the group consisting of: clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 9 systems and CRISPR/Cpf1 systems, wherein the at least one gene editor system effects expression of at least one immunomodulatory composition on a cell surface of a plurality of cells in a second TIL population, inhibiting expression of PD-1 and LAG-3. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

TALE method

The method for amplifying a TIL into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., process 2A) or as described in WO2018081473, WO2018129332, or WO2018182817, wherein the method further comprises gene editing at least a portion of the TIL by the TALE method. According to particular embodiments, use of the TALE method during the TIL amplification process may cause expression of at least one immunomodulatory composition at the cell surface, optionally causing silencing or reduction of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, use of the TALE method during the TIL amplification procedure may cause expression of at least one immunomodulatory composition at the cell surface, optionally causing enhancement of expression of one or more immune checkpoint genes in at least a portion of a therapeutic TIL population.

TALEs represent "transcription activator-like effector" proteins, which include TALENs ("transcription activator-like effector nucleases"). The method of gene editing using the TALE system is also referred to herein as the TALE method. TALE is a naturally occurring protein from the plant pathogenic bacterium Xanthomonas (Xanthomonas) that contains a DNA binding domain consisting of a series of repeat domains each recognizing a single base pair of 33 to 35 amino acids. TALE specificity is determined by two hypervariable amino acids called Repeated Variable Diradicals (RVDs). Modular TALE repeats are linked together to recognize a contiguous DNA sequence. Specific RVDs in the DNA binding domain recognize bases in the target locus, providing structural features to assemble a predictable DNA binding domain. The DNA binding domain of TALE is fused to the catalytic domain of a fokl endonuclease type IIS to prepare a targetable TALE nuclease. To induce site-specific mutations, two separate TALEN arms separated by a 14 to 20 base pair spacer region draw the fokl monomer closer to dimerize and create a targeted double strand break.

Several large systematic studies using various assembly methods indicate that TALE repeat sequences can be incorporated to identify virtually any user-defined sequence. Strategies that enable rapid assembly of custom TALE arrays include Golden Gate molecular cloning, high throughput solid phase assembly, and non-ligation dependent cloning techniques. Custom designed TALE arrays are also commercially available from CELLECTIS BIORESEARCH (paris, france), transposagen Biopharmaceuticals (lekurd, kentucky, usa) and Life Technologies (gland island, new york, usa). In addition, network-based tools (e.g., TAL effector-nucleotide target 2.0 (TAL Effector-Nucleotide Target 2.0)) can be used that are capable of designing custom TAL effector repeat arrays for the desired target and providing predicted TAL effector binding sites. See Doyle et al, nucleic acids research (Nucleic ACIDS RESEARCH), 2012, volume 40, W117-W122. Examples of TALE and TALEN processes suitable for use in the present invention are described in U.S. patent application publication nos. US2011/0201118 A1, US 2013/0177869 A1, US2013/0315884 A1, US2015/0203871 A1 and US 2016/012596 A1, the disclosures of which are incorporated herein by reference.

According to some embodiments of the invention, the TALE method comprises silencing or reducing expression of one or more immune checkpoint genes by inhibiting or preventing transcription of the target gene. For example, a TALE method can comprise utilizing KRAB-TALE, wherein the method comprises fusing a transcribed Kruppel-associated cassette (KRAB) domain with a DNA binding domain targeting a transcription start site of a gene, resulting in inhibiting or preventing transcription of the gene.

According to other embodiments, the TALE method comprises silencing or reducing the expression of one or more immune checkpoint genes by introducing mutations in the target gene. For example, the TALE method may comprise fusing a nuclease effector domain (e.g., fokl) to a TALE DNA binding domain, resulting in a TALEN. Fokl is active as a dimer; thus, the method includes constructing a TALEN pair to localize FOKL nuclease domains to adjacent genomic target sites, where the domains introduce DNA double-strand breaks. Double strand breaks can be completed after Fokl is correctly located and dimerized. After introduction of the double strand break, DNA repair can be achieved by two different mechanisms: high fidelity homologous recombination pair (HRR) (also known as homology directed repair or HDR) or error-prone non-homologous end joining (NHEJ). Repair of double strand breaks by NHEJ preferably results in deletions, insertions or substitutions of DNA target sites, i.e., NHEJ typically results in the introduction of small insertions and deletions at the break site, typically inducing a frameshift of knockout gene function. According to particular embodiments, the TALEN is directed to a majority of the 5' exons of the target gene, facilitating early frameshift mutations or premature stop codons. The gene mutation introduced by TALEN is preferably permanent. Thus, according to some embodiments, the method comprises silencing or reducing expression of an immune checkpoint gene by inducing a site-specific double-strand break by an error-prone NHEJ repair by utilizing the dimerized TALENs to cause one or more mutations in the target immune checkpoint gene.

According to other embodiments, TALENs are used to introduce genetic changes, such as non-random point mutations, target deletions, or additions of DNA fragments, by HRR. The introduction of a DNA double strand break enables gene editing by homologous recombination in the presence of a suitable donor DNA. According to some embodiments, the method comprises co-delivering a dimerized TALEN and a donor plasmid carrying a locus-specific homology arm to induce a site-specific double strand break and integrate more than one transgene into DNA.

According to other embodiments, TALENs, which are hybrid proteins derived from fokl and AvrXa7, as disclosed in U.S. patent publication No. 2011/0201118, can be used in accordance with embodiments of the present invention. This TALEN retains recognition specificity for the target nucleotide of AvrXa7 and double-stranded DNA cleavage activity of fokl. Other TALENs with different recognition specificities can be prepared using the same method. For example, compact (compact) TALENs can be generated by engineering core TALE scaffolds with different RVD sets to alter DNA binding specificity as well as targeting specific single dsDNA target sequences. See U.S. patent publication No. 2013/0177869. The catalytic domain of choice may be linked to a scaffold to effect DNA processing, which may be engineered to ensure that when fused to the core TALE scaffold, the catalytic domain is capable of processing DNA in the vicinity of a single dsDNA target sequence. Peptide linkers can also be engineered to fuse catalytic domains to scaffolds, resulting in compact TALENs made from a single polypeptide chain, which need not be used to target dimerization of a specific single dsDNA sequence. The core TALE scaffold may also be modified by fusing a catalytic domain (which may be a TAL monomer) to its N-terminus, achieving the possibility that this catalytic domain may interact with another catalytic domain fused to another TAL monomer, thereby creating a catalytic entity that may process DNA in the vicinity of the target sequence. See U.S. patent publication No. 2015/0203871. This architecture allows targeting only one DNA strand, which is not an option for the classical TALEN architecture.

According to some embodiments of the invention, conventional RVDs may be used to produce TALENs capable of significantly reducing gene expression. In some embodiments, four RVDs (i.e., NI, HD, NN, and NG) are used to target adenine, cytosine, guanine, and thymine, respectively. These conventional RVDs can be used, for example, to generate TALENs that target the PD-1 gene. Examples of TALENs using conventional RVDs include Gautron et al, molecular therapy: nucleic Acids (Molecular Therapy: nucleic Acids), 12 months 2017, volumes 9: 312-321 (Gautron), T3v1 and T1 TALENs disclosed herein are incorporated by reference. T3v1 and T1 TALENs target the second exon of the PDCD1 locus where the PD-L1 binding site is located and are capable of significantly reducing PD-1 production. In some embodiments, the T1 TALEN is synthesized by using the target SEQ ID NO:256, T3v1 TALEN by using the target SEQ ID NO:257 to achieve this.

According to other embodiments, the TALENs are modified using non-conventional RVDs to improve their activity and specificity for a target gene, such as disclosed in Gautron. Naturally occurring RVDs cover only a small portion of the potential diversity spectrum of hypervariable amino acid positions. Unconventional RVDs provide alternatives to natural RVDs and have novel inherent targeting specificity characteristics that can be used to exclude TALEN-targeted off-site targets (sequences that contain few mismatches relative to the target sequence in the genome). Non-conventional RVDs can be identified by generating and screening a collection of TALENs containing a combination of alternative amino acids at two hypervariable amino acid positions at a given position of the array, as disclosed in Juillerat et al, science report (SCIENTIFIC REPORTS) 5, no. 8150 (2015), incorporated herein by reference. Then, an unconventional RVD can be selected that is capable of distinguishing between the nucleotides present at the mismatched positions, which can prevent TALEN activity at the off-site sequence, while still allowing for proper processing of the target position. The selected non-conventional RVD may then be used to replace the conventional RVD in the TALEN. Examples of TALENs in which the regular RVD has been replaced by an irregular RVD include T3v2 and T3v3 PD-1 TALENs produced by Gautron. These TALENs have increased specificity compared to TALENs using conventional RVDs.

According to other embodiments, TALENs may be used to introduce gene changes to silence or reduce expression of both genes. For example, two independent TALENs can be generated to target two different genes and then used together. Molecular events generated by both TALENs at their respective loci and potentially off-target sites can be characterized by high throughput DNA sequencing. This allows for analysis of off-target sites and recognition of sites that may result from the use of two TALENs. Based on this information, appropriate conventional and non-conventional RVDs can be selected to engineer TALENs with increased specificity and activity even when used together. For example, gautron discloses the combined use of T3v4 PD-1 and TRAC TALEN to generate dual knockout CAR T cells that retain potent in vitro anti-tumor function.

In some embodiments, TILs may be genetically edited using the method of Gautron or other methods described herein, and then amplified by any of the procedures described herein. In some embodiments, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises the steps of:

(a) Activating a first TIL population obtained from a tumor resected from a patient using CD3 and CD28 activating beads or antibodies for 1 to 5 days;

(b) Gene editing at least a portion of the first population of TILs using electroporation of transcription activator-like effector nuclease-encoding nucleic acids to obtain a second population of TILs, wherein the gene editing effects expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of at least one immune checkpoint protein in a portion of cells of the second population of TILs;

(c) Optionally, culturing a second TIL population;

(d) Performing a first amplification by culturing the second population of TILs in a cell culture medium comprising IL-2 and optionally OKT-3, resulting in a third population of TILs, wherein the first amplification is performed in a closed container providing a first gas permeable surface area, the first amplification being performed for about 3 to 14 days to obtain the third population of TILs;

(e) Performing a second amplification by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs), resulting in a fourth TIL population, wherein the second amplification is performed for about 7 to 14 days to obtain the fourth TIL population, the fourth TIL population being a therapeutic TIL population;

(f) Collecting the therapeutic TIL population obtained from step (e);

(g) Transferring the collected TIL population from step (e) to an infusion bag, wherein the transferring of steps (e) to (f) is performed without opening the system; and

(H) Wherein one or more of steps (a) to (g) are performed in a closed, sterile system.

In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

In some embodiments, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises the steps of:

(b) Gene editing at least a portion of the first population of TILs using electroporation of transcription activator-like effector nuclease-encoding nucleic acids in a cell poration (cytoporation) medium to obtain a second population of TILs, wherein the gene editing effects expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of at least one immune checkpoint protein in a portion of cells of the second population of TILs;

(c) Optionally, culturing a second TIL population;

(d) Performing a first amplification by culturing the second population of TILs in a cell culture medium comprising IL-2 and optionally OKT-3, resulting in a third population of TILs, wherein the first amplification is performed in a closed container providing a first gas permeable surface area, the first amplification being performed for about 6 to 9 days to obtain the third population of TILs;

(e) Performing a second amplification by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a fourth TIL population, wherein the second amplification is performed for about 9 to 11 days to obtain the fourth TIL population, the fourth TIL population being a therapeutic TIL population;

(f) Collecting the therapeutic TIL population obtained from step (e);

(b) Gene editing at least a portion of the first population of TILs in a cell poration medium using electroporation of transcription activator-like effector nuclease encoding nucleic acids to obtain a second population of TILs, wherein the gene editing effects expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of at least one immune checkpoint protein in a portion of cells of the second population of TILs;

(c) Optionally, culturing the second TIL population, wherein culturing is performed at about 30 to 40 ℃ and about 5% CO ₂;

(f) Collecting the therapeutic TIL population obtained from step (e);

(d) Gene editing at least a portion of the third population of TILs in a cell poration medium using electroporation of transcription activator-like effector nuclease encoding nucleic acids to obtain a fourth population of TILs, wherein the gene editing effects expression of at least one immunomodulatory composition at the cell surface;

(e) Optionally, culturing a fourth TIL population; and

(b) Digesting tumor tissue in an enzyme medium to produce a tumor digest;

(e) Gene editing at least a portion of the third population of TILs in a cell poration medium using electroporation of transcription activator-like effector nuclease encoding nucleic acids to obtain a fourth population of TILs, wherein the gene editing effects expression of at least one immunomodulatory composition at the cell surface;

(f) Optionally, culturing a fourth TIL population, wherein culturing is performed at about 30 to 40 ℃ and about 5% CO ₂; and

(G) Culturing the fourth population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3, and IL-2 for about 5 to 15 days, resulting in an expanded number of TILs.

(d) Gene editing the third population of TILs by temporarily disrupting cell membranes of the third population of TILs to effect transfer of transcription activator-like effector nuclease encoding nucleic acid into the third population of TILs to obtain a fourth population of TILs, wherein the gene editing effects expression of at least one immunomodulatory composition at the cell surface;

(e) Optionally, culturing a fourth TIL population; and

(F) Culturing the fourth population of TILs in a second cell culture medium comprising Antigen Presenting Cells (APCs), OKT-3, and IL-2 for about 5 to 15 days, resulting in an expanded number of TILs. In some embodiments, the cell membrane of the third TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform.

(e) Optionally, culturing a fourth TIL population, wherein culturing is performed at about 30 to 40 ℃ and about 5% CO ₂; and

In some embodiments, the step of culturing the fourth TIL population is performed by: culturing the fourth population of TILs in the second cell culture medium for a first period of about 1 to 7 days, dividing the culture into a plurality of subcultures at the end of the first period, culturing each of the plurality of subcultures in a third medium comprising IL-2 for a second period of about 3 to 7 days, and combining the plurality of subcultures at the end of the second period to obtain an amplified number of TILs.

According to other embodiments, TALENs may be specifically designed to achieve a higher incidence of DSB events within target cells capable of targeting a specifically selected gene. See U.S. patent publication No. 2013/0315884. The use of such rare-cutting (rare cutting) endonucleases can increase the chance of achieving dual inactivation of target genes in transfected cells, enabling the production of engineered cells, such as T cells. In addition, other catalytic domains can be introduced with TALENs to increase mutation induction and enhance target gene inactivation. TALENs described in U.S. patent publication No. 2013/0315884 were successfully used to engineer T cells to make them suitable for immunotherapy. TALENs can also be used to inactivate various immune checkpoint genes in T cells, including inactivating at least two genes in a single T cell. See U.S. patent publication 2016/0125906. In addition, TALENs can be used to inactivate target genes encoding immunosuppressants and T cell receptors, as disclosed in U.S. patent publication No. 2018/0021379, which is incorporated herein by reference. Furthermore, TALENs may be used to inhibit expression of β2-microglobulin (B2M) and/or class II histocompatibility complex transactivator (CIITA), as disclosed in U.S. patent publication No. 2019/0010514, which is incorporated herein by reference.

Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of the TIL by TALE methods include PD-1、CTLA-4、LAG-3、HAVCR2(TIM-3)、Cish、TGFβ、PKA、CBL-B、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、BTLA、CD160、TIGIT、TET2、CD96、CRTAM、LAIR1、SIGLEC7、SIGLEC9、CD244、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT1、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2、GUCY1B3、TOX、SOCS1、ANKRD11 and BCOR.

Non-limiting examples of TALE-nucleases targeting the PD-1 gene are provided in the table below. In these examples, the target genomic sequence contains two 17-base pair (bp) long sequences (termed half-targets, shown in uppercase letters) separated by a 15-bp spacer (shown in lowercase letters). Each half target is recognized by a repeat sequence of a half TALE-nuclease listed in table 11. Thus, according to a specific embodiment, a TALE-nuclease of the invention recognizes and cleaves a target sequence selected from the group consisting of: SEQ ID NO:286 and SEQ ID NO:287.TALEN sequences and gene editing methods are also described in Gautron, supra.

Table 11: TALEN PD-1 sequence.

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(b) Gene editing at least a portion of a first population of TILs, wherein the gene editing comprises obtaining a second population of TILs in a cell poration medium using electroporation of a transcription activator-like effector nuclease encoding nucleic acid targeted to PD-1, the gene editing effecting expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of PD-1 in a portion of cells of the second population of TILs;

(d) Performing a first amplification by culturing a second population of TILs in a cell culture medium comprising IL-2 and optionally OKT-3, resulting in a third population of TILs, wherein the first amplification is performed in a closed container providing a first gas permeable surface area, wherein the first amplification is performed for about 6 to 9 days to obtain the third population of TILs;

(f) Collecting the therapeutic TIL population obtained from step (e);

(b) Gene editing at least a portion of the third population of TILs, wherein the gene editing comprises obtaining a fourth population of TILs in a cell poration medium using electroporation of a transcription activator-like effector nuclease encoding nucleic acid targeted to PD-1, the gene editing effecting expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of PD-1 in a portion of cells of the third population of TILs;

(d) Gene editing at least a portion of the third TIL population by temporarily disrupting cell membranes of the third TIL population to effect transfer of a transcription activator-like effector nuclease encoding nucleic acid targeting PD-1 into the third TIL population to obtain a fourth TIL population, wherein the gene editing effects expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of PD-1 in a portion of cells of the third TIL population;

In some embodiments, the cell membrane of the third TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Gene editing at least a portion of the first TIL population, wherein the gene editing comprises using targeting SEQ ID NO:149 or SEQ ID NO:150 to obtain a second population of TILs, the gene editing effecting expression of at least one immunomodulatory composition at the cell surface, inhibiting expression of PD-1 in a portion of cells of the second population of TILs;

(f) Collecting the therapeutic TIL population obtained from step (e);

(d) Gene editing at least a portion of a third TIL population, wherein the gene editing comprises using targeting SEQ ID NO:149 or SEQ ID NO:150 to obtain a fourth population of TILs, the gene editing effecting expression of at least one immunomodulatory composition at the cell surface, inhibiting expression of PD-1 in a portion of cells of the third population of TILs;

In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(d) Genetically editing at least a portion of the third TIL population by temporarily disrupting the cell membrane of the third TIL population to achieve targeting of the polypeptide of SEQ ID NO:149 or SEQ ID NO:150 into a third TIL population, to obtain a fourth TIL population, wherein gene editing effects expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of PD-1 in a portion of cells of the third TIL population;

(b) Gene editing at least a portion of the first TIL population, wherein the gene editing comprises using SEQ ID NO:157 and SEQ ID NO:158 or SEQ ID NO:153 and SEQ ID NO:154 to obtain a second population of TILs, the gene editing effecting expression of at least one immunomodulatory composition at the cell surface, inhibiting expression of PD-1 in a portion of cells of the second population of TILs;

(f) Collecting the therapeutic TIL population obtained from step (e);

(d) Gene editing at least a portion of the third TIL population, wherein the gene editing comprises using SEQ ID NO:157 and SEQ ID NO:158 or SEQ ID NO:153 and SEQ ID NO:154 to obtain a fourth population of TILs, the gene editing effecting expression of at least one immunomodulatory composition at the cell surface, inhibiting expression of PD-1 in a portion of the cells of the third population of TILs;

(d) Gene editing of at least a portion of the third TIL population by temporarily disrupting the cell membrane of the third TIL population, effecting the expression of SEQ ID NO:157 and SEQ ID NO:158 or SEQ ID NO:153 and SEQ ID NO:154 into a third TIL population, to obtain a fourth TIL population, wherein gene editing effects expression of at least one immunomodulatory composition at a cell surface, inhibiting expression of PD-1 in a portion of cells of the third TIL population;

Other non-limiting examples of genes that can be enhanced by permanent gene editing of TIL by the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CXCR 1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering expression of a target gene sequence by the TALE method and which may be used in accordance with embodiments of the present invention are described in U.S. patent No. 8,586,526, which is incorporated herein by reference. Examples of these disclosures include the use of non-naturally occurring DNA binding polypeptides having more than two TALE-repeat units containing a repeat RVD, an N-cap polypeptide made from residues of a TALE protein, and a C-cap polypeptide made from a fragment of the full-length C-terminal region of a TALE protein.

Examples of TALEN design and design strategies, activity assessment, screening strategies and Methods that can be used to effectively perform TALEN-mediated gene integration and inactivation are described in Valton et al, methods, 2014,69,151-170, which are incorporated herein by reference.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein the electroporation step comprises delivering a TALE nuclease system that effects expression of at least one immune modulating composition at the cell surface of a plurality of cells of the second TIL population that modulates expression of at least one immune checkpoint protein and/or adhesion molecule. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(e) Temporarily disrupting the cell membrane of the second TIL population to effect transfer of the at least one gene editor into the plurality of cells in the second TIL population;

(f) Standing the second TIL population for about 1 day;

Wherein the step of transferring the at least one gene editor comprises delivering a TALE nuclease system that effects expression of the at least one immune modulating composition at the cell surface of a plurality of cells of the second TIL population, modulating expression of the at least one immune checkpoint protein and/or adhesion molecule. In some embodiments, the cell membrane of the third TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(e) Performing a sterile electroporation step on the second population of TILs to effect transfer of the at least one gene editor into a plurality of cells in the second population of TILs;

(f) Standing the second TIL population for about 1 day;

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

Wherein the electroporation step comprises delivering a TALE nuclease system that effects expression of at least one immunomodulatory composition at a cell surface of a plurality of cells of the second TIL population, inhibiting expression of PD-1 and LAG-3. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein the step of transferring the at least one gene edit comprises delivering a TALE nuclease system that effects expression of the at least one immunomodulatory composition at a cell surface of a plurality of cells of the second TIL population, inhibiting expression of PD-1 and LAG-3. In some embodiments, the cell membrane of the third TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

C. Zinc finger method

The method for amplifying a TIL into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., process 2A) or as described in PCT/US2017/058610, PCT/US 2018/01605, or PCT/US2018/012633, wherein the method further comprises genetically editing at least a portion of the TIL by a zinc finger or zinc finger nuclease method. According to particular embodiments, the use of zinc finger methods during the TIL amplification process may cause expression of at least one immunomodulatory composition at the cell surface, optionally causing silencing or reduction of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of zinc finger methods during the TIL amplification process may cause expression of at least one immunomodulatory composition at the cell surface, optionally causing enhancement of expression of one or more immune checkpoint genes in at least a portion of a therapeutic TIL population.

The individual zinc fingers contain about 30 amino acids in a conserved ββα configuration. Several amino acids on the alpha-helical surface typically contact 3bp in the major groove of DNA at different levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA binding domain that includes eukaryotic transcription factors and contains zinc fingers. The second domain is a nuclease domain, which includes a fokl restriction enzyme and is responsible for catalytic cleavage of DNA.

The DNA binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and each can recognize between 9 and 18 base pairs. If the zinc finger domain is specific for its intended target site, even a pair of 3-finger ZFNs recognizing a total of 18 base pairs can theoretically target a single locus in the mammalian genome. One approach to creating new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The most common module assembly process involves combining three separate zinc fingers, each recognizing a3 base pair DNA sequence, to produce a3 finger array that recognizes 9 base pair target sites. Alternatively, a selection-based approach, such as oligo pool engineering (oligomerized pool engineering; OPEN), may be used to select new zinc finger arrays from random libraries that take into account background-dependent interactions between adjacent fingers (context-DEPENDENT INTERACTION). The engineered zinc fingers are commercially available; sangamo Biosciences (Richmong, california, U.S. has developed a specialized platform for zinc finger construction in cooperation with Sigma Aldrich (St. Louis, mitsui, U.S. A)

Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TIL by zinc finger methods include PD-1、CTLA-4、LAG-3、HAVCR2(TIM-3)、Cish、TGFβ、PKA、CBL-B、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、BTLA、CD160、TIGIT、CD96、CRTAM、LAIR1、SIGLEC7、SIGLEC9、CD244、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT1、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2、GUCY1B3、TOX、SOCS1、ANKRD11 and BCOR.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TIL by zinc finger methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions that alter expression of a target gene sequence by zinc finger methods and that can be used in accordance with embodiments of the present invention are described below: U.S. Pat. nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, which are incorporated herein by reference.

Other examples of altering expression of target gene sequences by zinc finger methods and systems, methods, and compositions that can be used in accordance with embodiments of the present invention are described in Beane et al, molecular therapy 2015,23 1380-1390, the disclosure of which is incorporated herein by reference.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein the electroporation step comprises delivering a zinc finger nuclease system that effects expression of at least one immunomodulatory composition at a cell surface of a plurality of cells of the second TIL population and modulating expression of at least one immune checkpoint protein and/or adhesion molecule. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein the step of transferring the at least one gene editor comprises delivering a zinc finger nuclease system that effects expression of the at least one immune modulating composition at the cell surface of the plurality of cells of the second TIL population and modulating expression of the at least one immune checkpoint protein and/or adhesion molecule. In some embodiments, the cell membrane of the third TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein the electroporation step comprises delivering a zinc finger nuclease system that effects expression of at least one immunomodulatory composition at a cell surface of a plurality of cells of the second TIL population, inhibiting expression of PD-1 and LAG-3. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

(b) Adding tumor fragments into the closed system;

(f) Standing the second TIL population for about 1 day;

Wherein the step of transferring the at least one gene edit comprises delivering a zinc finger nuclease system that effects expression of the at least one immunomodulatory composition at a cell surface of a plurality of cells of the second TIL population, inhibiting expression of PD-1 and LAG-3. In some embodiments, the cell membrane of the third TIL population is temporarily disrupted using a microfluidic platform. In some embodiments, the microfluidic platform is a SQZ carrier-free microfluidic platform. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed by: culturing the third or fourth TIL population or performing the second amplification for a first period of about 1 to 7 days, at the end of the first period, splitting the culture into a plurality of subcultures, culturing each of the plurality of subcultures with additional IL-2 for a second period of about 3 to 7 days, and at the end of the second period, combining the plurality of subcultures to obtain an amplified number of TILs or therapeutic TIL populations.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the first TIL population or the first amplification step is performed for about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 4 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 5 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 6 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 7 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 8 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 3 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 3 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 3 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 3 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 3 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 4 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 4 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 4 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 5 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 5 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 5 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 6 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 6 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 7 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 4 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first TIL population or the first amplification step is performed for about 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first TIL population or the first amplification step is performed for about 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first TIL population or the first amplification step is performed for about 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first TIL population or the first amplification step is performed for about 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first TIL population or the first amplification step is performed for about 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first TIL population or the first amplification step is performed for about 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the step of culturing the first TIL population or the first amplification step is performed for about 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the first TIL population or the first amplification step is performed for about 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 1, 2, 3, 4, 5, 6 or 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 2 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 3 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 4 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 5 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 6 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 1 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 1 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 1 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 1 to 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 1 to 2 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 2 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 3 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 4 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 5 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 3 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 3 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 2 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 2 to 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 2 to 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the activating step is performed for about 4 to 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the activating step is performed for about 1 day.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the activating step is performed for about 2 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the activating step is performed for about 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the activating step is performed for about 4 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the activating step is performed for about 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the activating step is performed for about 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the activating step is performed for about 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the stimulating step is performed for about 1, 2, or 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the stimulating step is performed for about 1 to 2 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the stimulating step is performed for about 2 to 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the stimulating step is performed for about 1 day.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the stimulating step is performed for about 2 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, such that the stimulating step is performed for about 3 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 9 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 10 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 11 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 12 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 13 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 14 to 15 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 9 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 10 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 11 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 12 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 13 to 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 to 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 to 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 to 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 to 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 9 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 9 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 9 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 9 to 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 10 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 10 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 10 to 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 11 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 11 to 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 12 to 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 6 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 7 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 8 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 9 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 10 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 11 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 12 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 13 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 14 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above such that the step of culturing the third or fourth TIL population or the second amplification step is performed for about 15 days.

V. examples of methods of expanding therapeutic T cells including Peripheral Blood (PBL) and/or bone Marrow (MIL)

A. Method for amplifying Peripheral Blood Lymphocytes (PBLs) derived from peripheral blood

PBL method 1. In some embodiments of the invention, the PBLs are amplified using the methods described herein. In some embodiments of the invention, the method comprises obtaining a PBMC sample from whole blood. In some embodiments, the method comprises enriching T cells by using negative selection of non-cd19+ fractions to isolate pure T cells from PBMCs. On day 0, pure T cells were compared to anti-CD 3/anti-CD 28 antibodies at a 1:1 ratio (beads: cells)IL-2 at 3000IU/mL was cultured together. On day 4, 3000IU/mL of additional IL-2 was added to the culture. On day 7, anti-CD 3/anti-CD 28 antibody was reusedThe cultures were stimulated at a ratio of 1:1 (beads: cells), and 3000IU/mL of additional IL-2 was added to the cultures. PBLs were collected on day 14, beads removed and PBLs were counted and phenotyped. In some embodiments, the method comprises enriching T cells by using bead-based negative selection of non-cd19+ fractions to isolate pure T cells from PBMCs.

In some embodiments of the invention, PBL method 1 proceeds as follows: on day 0, cryopreserved PBMC samples were thawed and PBMC numbers were counted. T cells were isolated using the human pan T cell isolation kit with LS column (meitian gentle biotechnology). Isolated T cells were counted and seeded at 5 x 10 ⁵ cells/well in GRex well plates and at a 1:1 ratio toTogether (anti-CD 3/anti-CD 28) and 3000IU/mL IL-2 were co-cultured in a total of 8mL of CM2 medium per well. On day 4, the medium in each well was changed from CM2 to AIM-V with fresh 3000IU/mL IL-2. On day 7, the expanded cells were collected, counted, and then 15X 10 ⁶ cells per bottle in GRex I M flasks at a 1:1 ratio (beads: cells) to/>Together with 3000IU/mL IL-2 in a total of 100mL AIM-V medium. On day 11, the medium was replaced with CM-4 medium supplemented with fresh 3000IU/mL IL-2. On day 14, the removal/>, was performed using DynaMag magnets (DynaMag ^TM -15)Cells were counted.

In some embodiments of the invention, PBL method 1 proceeds as follows: on day 0, cryopreserved PBMC samples were thawed and PBMC numbers were counted. T cells were isolated using the human pan T cell isolation kit with LS column (meitian gentle biotechnology). Isolated T cells were counted and seeded at 5 x 10 ⁵ cells/well in GRex well plates at a ratio of 1:1 toTogether (anti-CD 3/anti-CD 28) and 3000IU/mL IL-2 were co-cultured in a total of 8mL of CM2 medium per well. On day 4, the medium in each well was changed from CM2 to AIM-V with fresh 3000IU/mL IL-2. On day 7, PBLs were collected, counted, and then plated in new GRex-24 well plates at 1X 10 ⁶ cells/well at a 1:1 ratio (beads: cells) to/>Together with 3000IU/mL IL-2 in a total of 8mL AIM-V medium. On day 11, the medium was replaced with CM-4 medium supplemented with fresh 3000IU/mL IL-2. On day 14, the removal/>, was performed using DynaMag magnets (DynaMag ^TM -15)And the cells were counted.

PBL method 2. In some embodiments of the invention, the PBLs are amplified using PBL method 2, which includes obtaining PBMC samples from whole blood. T cells from PBMCs were enriched by culturing PBMCs at 37 ℃ for at least three hours and then isolating non-adherent cells. Non-adherent cells were expanded similarly to PBL method 1, i.e., on day 0, non-adherent cells were compared to anti-CD 3/anti-CD 28 antibodies at a ratio of 1:1 (beads: cells)And 3000IU/mL IL-2. On day 4, 3000IU/mL of additional IL-2 was added to the culture. On day 7, anti-CD 3/anti-CD 28 antibody/>Cultures were stimulated at a 1:1 ratio (beads: cells) and 3000IU/mL of additional IL-2 was added to the cultures. PBLs were collected on day 14, beads removed and PBLs were counted and phenotyped. /(I)

In some embodiments of the invention, PBL method 2 proceeds as follows: on day 0, the cryopreserved PMBC samples were thawed and PBMC cells were seeded at 6 million cells per well in CM-2 medium in 6-well plates and incubated for 3 hours at 37 ℃. After 3 hours, non-adherent cells (which are PBLs) were removed and counted. PBLs were combined with anti-CD 3/anti-CD 28 at a 1:1 bead to cell ratio in each well of GRex24 well plates at 1 x 10 ⁶ cells/wellTogether with 3000IU/mL IL-2 in a total of 7mL CM-2 medium. On day 4, the medium in each well was replaced with AIM-V medium and fresh 3000IU/mL IL-2. On day 7, the expanded cells were collected, counted, and then 15X 10 ⁶ cells per bottle in GRex I M flasks at a 1:1 ratio (T cells: beads) to/>Together with 3000IU/mL IL-2 in a total of 100mL AIM-V medium. On day 11, the medium was replaced with CM-4 medium and fresh IL-2 (3000 IU/mL) was supplemented. On day 14, dynaBeads were removed and cells were counted using DynaMag ^TM magnets (DynaMag ^TM -15).

In some embodiments of the invention, PBL method 2 proceeds as follows: on day 0, the cryopreserved PMBC samples were thawed and PBMC cells were seeded at 6 million cells per well in CM-2 medium in 6-well plates and incubated for 3 hours at 37 ℃. After 3 hours, non-adherent cells (which are PBLs) were removed and counted. PBLs were combined with anti-CD 3/anti-CD 28 at a 1:1 bead to cell ratio in each well of GRex24 well plates at 1 x 10 ⁶ cells/wellTogether with 3000IU/mL IL-2 in a total of 7mL CM-2 medium. On day 4, the medium in each well was replaced with AIM-V medium and fresh 3000IU/mL IL-2. On day 7, the expanded cells were collected, counted, and then plated in a new GRex well plate at 1X 10 ⁶ cells/well at a 1:1 ratio (T cells: beads) to/>Together with 3000IU/mL IL-2 in a total of 8mL AIM-V medium. On day 11, the medium was replaced with CM-4 medium and fresh IL-2 (3000 IU/mL) was supplemented. On day 14, dynaBead was removed and cells were counted using DynaMag ^TM magnets (DynaMag ^TM -15).

PBL method 3. In some embodiments of the invention, the PBLs are amplified using PBL method 3, which includes obtaining PBMC samples from peripheral blood. B cells were isolated using cd19+ selection and T cells were selected using non-cd19+ fractions of negative selection PBMC samples. On day 0, T cells and B cells were combined with anti-CD 3/anti-CD 28 antibodies at a 1:1 ratio (beads: cells)And 3000IU/mL IL-2 co-culture. On day 4, 3000IU/mL of additional IL-2 was added to the culture. On day 7, anti-CD 3/anti-CD 28 antibody/>The cultures were stimulated at a ratio of 1:1 (beads: cells), and 3000IU/mL of additional IL-2 was added to the cultures. PBLs were collected on day 14, beads removed and PBLs were counted and phenotyped.

In some embodiments of the invention, PBL method 3 proceeds as follows: on day 0, cryopreserved PBMCs from peripheral blood were thawed and counted. Cd19+ B cells were sorted using the human CD19 Multisort kit (meitian gentle biotechnology). In the non-cd19+ cell fraction, T cells were purified using a human pan T cell isolation kit and LS column (meitian gentle biotechnology). T cells (PBLs) and B cells were co-cultured at different rates in about 8ml of CM2 medium in Grex's 24 well plates in the presence of about 3000IU/ml IL-2. The ratio of B cells to T cells was 0.1:1, 1:1 and 10:1. With anti-CD 3/anti-CD 28 antibodiesT cell/B cell co-cultures were stimulated at a ratio of 1:1 (beads: cells). On day 4, the medium was changed from CM2 to AIM-V medium and 3000IU/mL of additional IL-2 was added to the culture. On day 7, cells were collected and counted, re-inoculated in AIM-V medium in a range of about 1.5X10 ⁵ to about 4X 10 ⁵ cells/well in a new Grex-24 well plate with anti-CD 3/anti-CD 28 antibody/>, at a ratio of 1:1 (beads: cells)And additional IL-2 stimulation of 3000 IU/ml. On day 14, dynaBead was removed using DynaMag ^TM magnets (DynaMag ^TM -15) and the cells were counted.

In some embodiments, the PBMCs are isolated from a whole blood sample. In some embodiments, PBMC samples are used as starting material for amplifying PBLs. In some embodiments, the sample is cryopreserved prior to the amplification process. In other embodiments, fresh samples are used as starting materials for the amplification of PBLs. In some embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human pan T cell isolation kit and LS column. In some embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art (e.g., CD19 negative selection).

In some embodiments of the invention, the process proceeds for about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In other embodiments, the process is performed for about 7 days. In other embodiments, the process is performed for about 14 days.

In some embodiments of the invention, PBMCs are incubated with an anti-CD 3/anti-CD 28 antibody. In some embodiments, any useful anti-CD 3/anti-CD 28 product may be used in the present invention. In some embodiments of the invention, the commercially available products used areIn some embodiments, will/>With PBMC at a ratio of 1:1 (beads: cells). In other embodiments, the antibody is/>, which is cultured with PBMCs at a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1 (beads: cells)In some embodiments of the invention, the antibody culturing step and/or the step of re-stimulating the cells with antibody is performed for a period of time of about 2 to about 6 days, about 3 to about 5 days, or about 4 days. In some embodiments of the invention, the antibody culturing step is performed for a period of about 2 days, 3 days, 4 days, 5 days, or 6 days.

In some embodiments, PBMC samples are incubated with IL-2. In some embodiments of the invention, the cell culture medium used to amplify the PBLs from the PBMCs comprises IL-2 at a concentration selected from the group consisting of: about 100IU/mL, about 200IU/mL, about 300IU/mL, about 400IU/mL, about 100IU/mL, about 500IU/mL, about 600IU/mL, about 700IU/mL, about 800IU/mL, about 900IU/mL, about about 1,000IU/mL, about 1,300IU/mL, about/L, about about 2,000IU/mL, about/mL about/mL, about 3,000IU/mL, about/L, about/mL, about/mL about/L, about/L about/mL, about/mL about/mL, about/mL.

In some embodiments of the invention, the starting cell number of the PBMCs used in the amplification process is from about 25,000 to about 1,000,000, from about 30,000 to about 900,000, from about 35,000 to about 850,000, from about 40,000 to about 800,000, from about 45,000 to about 800,000, from about 50,000 to about 750,000, from about 55,000 to about 700,000, from about 60,000 to about 650,000, from about 65,000 to about 600,000, from about 70,000 to about 550,000, preferably from about 75,000 to about 500,000, from about 80,000 to about 450,000, from about 85,000 to about 400,000, from about 90,000 to about 350,000, from about 95,000 to about 300,000, from about 100,000 to about 250,000, from about 105,000 to about 200,000, or from about 110,000 to about 150,000. In some embodiments of the invention, the starting cell number of the PBMCs is about 138,000, 140,000, 145,000 or more. In other embodiments, the starting cell number of PBMCs is about 28,000. In other embodiments, the starting cell number of PBMCs is about 62,000. In other embodiments, the starting cell number of PBMCs is about 338,000. In other embodiments, the starting cell number of PBMCs is about 336,000.

In some embodiments of the invention, cells are grown in GRex well plates. In some embodiments of the invention, similar well plates are used. In some embodiments, the starting material for expansion is about 5x 10 ⁵ T cells/well. In some embodiments of the invention, there are 1×10 ⁶ cells/well. In some embodiments of the invention, the number of cells per well is sufficient to inoculate the well and expand T cells.

In some embodiments of the invention, the amplification factor of the PBL is from about 20% to about 100%, 25% to about 95%, 30% to about 90%, 35% to about 85%, 40% to about 80%, 45% to about 75%, 50% to about 100%, or 25% to about 75%. In some embodiments of the invention, the amplification factor is about 25%. In other embodiments of the invention, the amplification factor is about 50%. In other embodiments, the amplification factor is about 75%.

In some embodiments of the invention, additional IL-2 may be added to the culture for one or more days throughout the process. In some embodiments of the invention, additional IL-2 is added on day 4. In some embodiments of the invention, additional IL-2 is added on day 7. In some embodiments of the invention, additional IL-2 is added on day 11. In another embodiment, additional IL-2 is added on day 4, day 7, and/or day 11. In some embodiments of the invention, the cell culture medium may be changed during one or more days of the cell culture process. In some embodiments, the cell culture medium is changed on day 4, day 7, and/or day 11 of the process. In some embodiments of the invention, the PBL is incubated with additional IL-2 for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments of the invention, the PBL is incubated for a period of 3 days after each addition of IL-2.

In some embodiments, the cell culture medium is replaced at least once during the method. In some embodiments, the cell culture medium is replaced while additional IL-2 is added. In other embodiments, the cell culture medium is changed on at least one of day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14. In some embodiments of the invention, the cell culture medium used during the entire process may be the same or different. In some embodiments of the invention, the cell culture medium is CM-2, CM-4 or AIM-V.

In some embodiments of the invention, T cells may be restimulated with anti-CD 3/anti-CD 28 antibodies for one or more days during the entire 14 day expansion process. In some embodiments, T cells are re-stimulated on day 7. In some embodiments, the restimulation step is performed using GRex M bottles. In some embodiments of the invention, similar bottles are used.

In some embodiments of the invention, dynaMag ^TM magnets are used for removalCells were counted and analyzed using the phenotypic and functional assays described further in the examples below. In some embodiments of the invention, the antibodies are isolated from the PBL or MILs using methods known in the art. In any of the foregoing embodiments, a bead-based selection of TIL, PBL, or MILs is used.

In some embodiments of the invention, the PBMC sample is cultured for a period of time and at a desired temperature effective to identify non-adherent cells. In some embodiments of the invention, the incubation time is about 3 hours. In some embodiments of the invention, the temperature is about 37 ℃. Non-adherent cells are then expanded using the procedure described above.

In some embodiments of the invention, PBMCs are obtained from a patient who has been treated with ibrutinib or another ITK or kinase inhibitor, such ITK and kinase inhibitor being as described elsewhere herein. In some embodiments of the invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and irreversibly binds ITK. In some embodiments of the invention, the ITK inhibitor is an allosteric ITK inhibitor that binds ITK. In some embodiments of the invention, the PBMCs are obtained from: the patient has been treated with ibrutinib or other ITK inhibitors (including ITK inhibitors as described elsewhere herein) prior to obtaining a PBMC sample for use with any of the foregoing methods (including PBL method 1, PBL method 2 or PBL method 3). In some embodiments of the invention, ITK inhibitor therapy has been administered at least 1, at least 2, or at least 3 or more times. In some embodiments of the invention, the lag3+, PD-1+ cells contained in PBLs expanded from patients that have been pre-treated with ibrutinib or other ITK inhibitors are less than PBLs expanded from patients that have not been pre-treated with ibrutinib or other ITK inhibitors. In some embodiments of the invention, the level of ifnγ production contained in PBLs amplified from patients that have been pretreated with ibrutinib or other ITK inhibitors is increased compared to PBLs amplified from patients that have not been pretreated with ibrutinib or other ITK inhibitors. In some embodiments of the invention, PBLs amplified from patients who have been treated with ibrutinib or other ITK inhibitors comprise increased lytic activity at lower effector to target cell ratios than PBLs amplified from patients who have not been pretreated with ibrutinib or other ITK inhibitors. In some embodiments of the invention, patients that have been pretreated with ibrutinib or other ITK inhibitors have a higher fold expansion than untreated patients.

In some embodiments of the invention, the method comprises the step of adding an ITK inhibitor to the cell culture. In some embodiments, the ITK inhibitor is added on one or more of day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 in the process. In some embodiments, the ITK inhibitor is added on the day of replacement of cell culture medium during the method. In some embodiments, the ITK inhibitor is added on day 0 and at the time of cell culture medium replacement. In some embodiments, the ITK inhibitor is added at the time of addition of IL-2 during the method. In some embodiments, the ITK inhibitor is added on day 0, day 4, day 7, and optionally day 11 of the method. In some embodiments of the invention, the ITK inhibitor is added on days 0 and 7 of the method. In some embodiments of the invention, the ITK inhibitor is an ITK inhibitor known in the art. In some embodiments of the invention, the ITK inhibitor is an ITK inhibitor as described elsewhere herein.

In some embodiments of the invention, the concentration of ITK inhibitor used in the method is about 0.1nM to about 5 μm. In some embodiments, the ITK inhibitor is used in the method at a concentration of about 0.1nM、0.5nM、1nM、5nM、10nM、20nM、30nM、40nM、50nM、60nM、70nM、80nM、90nM、100nM、150nM、200nM、250nM、300nM、350nM、400nM、450nM、500nM、550nM、600nM、650nM、700nM、750nM、800nM、850nM、900nM、950nM、1μM、2μM、3μM、4μM or 5 μm.

In some embodiments of the invention, the method comprises the step of adding an ITK inhibitor when PBMCs are derived from a patient not previously exposed to ITK inhibitor treatment (e.g., ibrutinib).

In some embodiments, the PBMC sample is from a subject or patient that has optionally been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor that has been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more. In other embodiments, the PBMCs are derived from a patient currently undergoing an ITK inhibitor regimen, such as ibutenib (ibrutinib).

In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and is refractory to treatment with the kinase inhibitor or ITK inhibitor (e.g., ibutinib).

In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor, but is no longer treated with a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor, but is no longer treated with the kinase inhibitor or the ITK inhibitor, and has not been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or longer. In other embodiments, the PBMCs are derived from patients previously exposed to the ITK inhibitor but have not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In some embodiments of the invention, on day 0, cells are selected for cd19+ and sorted accordingly. In some embodiments of the invention, the selection is performed using antibody binding beads. In some embodiments of the invention, the pure T cells are isolated from PBMCs on day 0. In some embodiments of the invention, cd19+ B cells and naive T cells are co-cultured with anti-CD 3/anti-CD 28 antibodies for at least 4 days on day 0. In some embodiments of the invention, IL-2 is added to the culture on day 4. In some embodiments of the invention, the culture is re-stimulated with anti-CD 3/anti-CD 28 antibodies and additional IL-2 on day 7. In some embodiments of the invention, PBLs are collected on day 14.

In some embodiments of the invention, for patients not pre-treated with ibrutinib or other ITK inhibitors, 10-15ml of Buffy Coat will produce about 5 x 10 ⁹ PBMCs, which in turn will produce about 5.5 x 10 ⁷ starting cell materials and about 11 x 10 ⁹ PBLs at the end of the amplification process. In some embodiments of the invention, about 54×10 ⁶ PBMCs will produce about 6×10 ⁵ starting materials and about 1.2×10 ⁸ MILs (about 205-fold amplification).

In some embodiments of the invention, the amplification process will yield about 20×10 ⁹ PBLs for patients pre-treated with ibrutinib or other ITK inhibitors. In some embodiments of the invention, 40.3X10 ⁶ PBMC will produce about 4.7X10 ⁵ starting cell material and about 1.6X10 ⁸ PBLs (about 338 fold expansion).

In some embodiments of the invention, the clinical dosages of PBLs of the invention that are applicable to Chronic Lymphocytic Leukemia (CLL) patients are about 0.1×10 ⁹ to about 15×10 ⁹ PBLs, about 0.1×10 ⁹ to about 15×10 ⁹ PBLs, about 0.12×10 ⁹ to about 12×10 ⁹ PBLs, about 0.15×10 ⁹ to about 11×10 ⁹ PBLs, about 0.2×10 ⁹ to about 10×10 ⁹ PBLs, about 0.3×10 ⁹ to about 9×10 ⁹ PBLs, about 0.4×10 ⁹ to about 8×10 ⁹ PBLs, about 0.5×10 ⁹ to about 7×10 ⁹ PBLs, about 0.6×10 ⁹ to about 6×10 PBLs, about 0.7×10 ⁹ to about 5×10 ⁹ PBLs, about 0.8×10 ⁹ to about 11×10 ⁹ PBLs, about 0.2×10 to about 2×10 ⁹ PBLs, about 2×10 ⁹ ×2 to about ⁹ ×10 pb ⁹.

In any of the foregoing embodiments, the PBMCs may be derived from whole blood samples, obtained by apheresis, derived from buffy coat, or obtained from any other method known in the art for obtaining PBMCs.

B. Method for amplifying bone Marrow Infiltrating Lymphocytes (MILs) from bone marrow derived PBMCs

MILs method 1. In some embodiments of the invention, methods of amplifying MILs from bone marrow derived PBMCs are described. In some embodiments of the invention, the method is performed for 14 days. In some embodiments, the method comprises obtaining bone marrow PBMCs and cryopreserving the PBMCs. On day 0, PBMC were combined with anti-CD 3/anti-CD 28 antibodies at a 1:1 ratio (beads: cells)And 3000IU/mL IL-2. On day 4, 3000IU/mL of additional IL-2 was added to the culture. On day 7, anti-CD 3/anti-CD 28 antibody/>The cultures were stimulated at a ratio of 1:1 (beads: cells), and 3000IU/mL of additional IL-2 was added to the cultures. MILs were collected on day 14, beads were removed, and MILs were optionally counted and phenotyped.

In some embodiments of the invention, MILs method 1 is performed as follows: on day 0, cryopreserved bone marrow-derived PBMC samples were thawed and PBMCs were counted. PBMCs were combined with anti-CD 3/anti-CD 28 antibodies at 5x 10 ⁵ cells/well in GRex-24 well plates in the presence of 3000IU/ml IL-2About 8ml of CM-2 cell culture medium (comprising RPMI-1640, human AB serum, L-glutamine, 2-mercaptoethanol, gentamicin sulfate (GENTAMICIN SULFATE), AIM-V medium) was co-cultured at a 1:1 ratio per well. On day 4, the cell culture medium was replaced with AIM-V supplemented with 3000IU/ml of additional IL-2. On day 7, the amplified MILs were counted. 1X 10 ⁶ cells/well were transferred into a new GRex well plate with an anti-CD 3/anti-CD 28 antibody/> in the presence of 3000IU/ml IL-2 in a ratio of 1:1Together in about 8ml AIM-V medium per well. On day 11, the cell culture medium was changed from AIM-V to CM-4 (containing AIM-V medium, 2mM Glutamax and 3000IU/mL IL 2). On day 14, remove/>, using DynaMag magnets (DynaMag ^TM 15)And counts MILs.

MILs method 2. In some embodiments of the invention, the method is performed for 7 days. In some embodiments, the method comprises obtaining bone marrow derived PBMCs and cryopreserving the PBMCs. On day 0, PBMC were combined with anti-CD 3/anti-CD 28 antibodies at a 3:1 ratio (beads: cells)And 3000IU/mL IL-2. MILs were collected on day 7, beads were removed, and MILs were optionally counted and phenotyped.

In some embodiments of the invention, MILs method 2 is performed as follows: on day 0, cryopreserved PBMC samples were thawed and PBMCs were counted. PBMCs were combined with anti-CD 3/anti-CD 28 antibodies at 5 x 10 ⁵ cells/well in GRex-24 well plates in the presence of 3000IU/ml IL-2About 8ml of CM-2 cell culture medium (comprising RPMI-1640, human AB serum, L-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium) was co-cultured at a 1:1 ratio per well. On day 7, remove/>, using DynaMag magnets (DynaMag ^TM 15)MILs are counted.

MILs method 3. In some embodiments of the invention, the method comprises obtaining PBMCs from bone marrow. On day 0, PBMCs were selected for cd3+/cd33+/cd20+/cd14+, sorted, non-cd3+/cd33+/cd20+/cd14+ cell fractions sonicated, and a portion of the sonicated cell fraction was added back to the selected cell fraction. 3000IU/ml IL-2 was added to the cell culture. On day 3, PBMC were combined with anti-CD 3/anti-CD 28 antibodies at a 1:1 ratio (beads: cells)And 3000IU/ml IL-2. On day 4, 3000IU/mL of additional IL-2 was added to the culture. On day 7, anti-CD 3/anti-CD 28 antibody/>The cultures were stimulated at a ratio of 1:1 (beads: cells), and 3000IU/mL of additional IL-2 was added to the cultures. On day 11, 3000IU/ml IL-2 was added to the culture. MILs were collected on day 14, beads removed, optionally counted and phenotyped.

In some embodiments of the invention, MILs method 3 proceeds as follows: on day 0, cryopreserved PBMC samples were thawed and PBMC numbers were counted. Cells were stained with CD3, CD33, CD20 and CD14 antibodies and sorted using an S3e cell sorter (Bio-Rad). Cells were sorted into two fractions: immune cell fraction (MILs fraction) (cd3+cd33+cd20+cd14+) and AML blast cell fraction (non-cd3+cd33+cd20+cd14+). Cells from the AML blast fraction, which are approximately equal in number to cells from the immune cell fraction (or MILs fraction) to be seeded on Grex well plates, were suspended in 100 μl of medium and sonicated. In this example, from about 2.8X10 ⁴ to about 3.38X10 ⁵ cells from the AML blast fraction were obtained, suspended in 100. Mu.l CM2 medium, and sonicated for 30 seconds. In Grex-24 well plates, 100 μl of sonicated AML blasts fraction was added to immune cells fraction. In the presence of 6000IU/ml IL-2, immune cells were present in about 8ml CM-2 cell culture medium per well in an amount of about 2.8X10 ⁴ to about 3.38X10 ⁵ cells/well, and were cultured with this fraction of AML embryo cells for about 3 days. On day 3, anti-CD 3/anti-CD 28 antibodies were raised againstAdded to each well at a ratio of 1:1 and incubated for about 1 day. On day 4, the cell culture medium was replaced with AIM-V supplemented with 3000IU/ml of additional IL-2. On day 7, the amplified MILs were counted. About 1.5X10 ⁵ to 4X 10 ⁵ cells/well were transferred into a new GRex well plate with an anti-CD 3/anti-CD 28 antibody/> in a ratio of 1:1 in the presence of 3000IU/ml IL-2Together in about 8ml AIM-V medium per well. On day 11, the cell culture medium was changed from AIM-V to CM-4 (supplemented with 3000IU/ml IL-2). On day 14, remove/>, using DynaMag magnets (DynaMag ^TM 15)MILs are optionally counted.

In some embodiments of the invention, the PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, needle aspiration of living tissue sections, or other similar means known in the art. In some embodiments, the PBMCs are fresh. In other embodiments, the PBMCs are cryopreserved.

In some embodiments of the invention, the method is performed for about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In other embodiments, the method is performed for about 7 days. In other embodiments, the method is performed for about 14 days.

In some embodiments of the invention, PBMCs are incubated with an anti-CD 3/anti-CD 28 antibody. In some embodiments, any useful anti-CD 3/anti-CD 28 product may be used in the present invention. In some embodiments of the invention, the commercially available products used areIn some embodiments, will/>With PBMC at a ratio of 1:1 (beads: cells). In other embodiments, the antibody is/>, which is cultured with PBMCs at a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1 (beads: cells)In any of the foregoing examples, a magnetic bead-based selection of immune cell fraction (or MILs fraction) (cd3+cd33+cd20+cd14+) or AML blast cell fraction (non-cd3+cd33+cd20+cd14+) was used. In some embodiments of the invention, the antibody culturing step and/or the step of re-stimulating the cells with antibody is performed for a period of time of about 2 to about 6 days, about 3 to about 5 days, or about 4 days. In some embodiments of the invention, the antibody culturing step is performed for a period of about 2 days, 3 days, 4 days, 5 days, or 6 days.

In some embodiments of the invention, the ratio of the number of cells from the AML blasts fraction to the number of cells from the immune cells fraction (or MILs fraction) is about 0.1:1 to about 10:1. In other embodiments, the ratio is about 0.1:1 to about 5:1, about 0.1:1 to about 2:1, or about 1:1. In some embodiments of the invention, AML blasts fraction is optionally destroyed to break cell aggregation. In some embodiments, the AML blasts fraction is destroyed using sonication, homogenization, cell lysis, vortexing, or shaking. In other embodiments, the AML blasts fraction is disrupted using sonication. In some embodiments of the invention, the non-cd3+, non-cd33+, non-cd20+, non-cd14+ cell fraction (AML blasts fraction) is lysed using suitable lysis methods, including pyrolysis, chemical lysis (e.g., organic alcohols), enzymatic lysis, and other cell lysis methods known in the art.

In some embodiments of the invention, cells from AML blasts fraction are suspended at a concentration of about 0.2 x 10 ⁵ to about 2 x 10 ⁵ cells per 100 μl, added to the cell culture along with immune cell fraction. In other embodiments, the concentration is about 0.5X10 ⁵ to about 2X 10 ⁵ cells per 100. Mu.L, about 0.7X10 ⁵ to about 2X 10 ⁵ cells per 100. Mu.L, about 1X 10 ⁵ to about 2X 10 ⁵ cells per 100. Mu.L, or about 1.5X10 ⁵ to about 2X 10 ⁵ cells per 100. Mu.L.

In some embodiments, PBMC samples are incubated with IL-2. In some embodiments of the invention, the cell culture medium used to amplify MILs comprises IL-2 at a concentration selected from the group consisting of: about 100IU/mL, about 200IU/mL, about 300IU/mL, about 400IU/mL, about 100IU/mL, about 500IU/mL, about 600IU/mL, about 700IU/mL, about 800IU/mL, about 900IU/mL, about about 1,000IU/mL, about 1,300IU/mL, about/L, about about 2,000IU/mL, about/mL about/mL, about 3,000IU/mL, about/L, about/mL, about/mL about/L, about/L about/mL, about/mL about/mL, about/mL.

In some embodiments of the invention, additional IL-2 may be added to the culture throughout one or more days of the process. In some embodiments of the invention, additional IL-2 is added on day 4. In some embodiments of the invention, additional IL-2 is added on day 7. In some embodiments of the invention, additional IL-2 is added on day 11. In other embodiments, additional IL-2 is added on day 4, day 7, and/or day 11. In some embodiments of the invention, MILs are incubated with additional IL-2 for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments of the invention, MILs are cultured for a period of 3 days after each addition of IL-2.

In some embodiments, the cell culture medium is replaced at least once during the method. In some embodiments, the cell culture medium is replaced while additional IL-2 is added. In other embodiments, the cell culture medium is changed on at least one of day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14. In some embodiments of the invention, the cell culture medium used during the entire process may be the same or different. In some embodiments of the invention, the cell culture medium is CM-2, CM-4 or AIM-V. In some embodiments of the invention, the cell culture media exchange step at day 11 is optional. In some embodiments of the invention, the starting cell number of the PBMCs used in the amplification process is from about 25,000 to about 1,000,000, from about 30,000 to about 900,000, from about 35,000 to about 850,000, from about 40,000 to about 800,000, from about 45,000 to about 800,000, from about 50,000 to about 750,000, from about 55,000 to about 700,000, from about 60,000 to about 650,000, from about 65,000 to about 600,000, from about 70,000 to about 550,000, preferably from about 75,000 to about 500,000, from about 80,000 to about 450,000, from about 85,000 to about 400,000, from about 90,000 to about 350,000, from about 95,000 to about 300,000, from about 100,000 to about 250,000, from about 105,000 to about 200,000, or from about 110,000 to about 150,000. In some embodiments of the invention, the starting cell number of the PBMCs is about 138,000, 140,000, 145,000 or more. In other embodiments, the starting cell number of PBMCs is about 28,000. In other embodiments, the starting cell number of PBMCs is about 62,000. In other embodiments, the starting cell number of PBMCs is about 338,000. In other embodiments, the starting cell number of PBMCs is about 336,000.

In some embodiments of the invention, MILs are amplified by about 20% to about 100%, 25% to about 95%, 30% to about 90%, 35% to about 85%, 40% to about 80%, 45% to about 75%, 50% to about 100%, or 25% to about 75%. In some embodiments of the invention, the amplification factor is about 25%. In other embodiments of the invention, the amplification factor is about 50%. In other embodiments, the amplification factor is about 75%.

In some embodiments of the invention, MILs are amplified from 10-50ml bone marrow aspirate. In some embodiments of the invention, 10ml bone marrow aspirate is obtained from a patient. In other embodiments, 20ml bone marrow aspirate is obtained from a patient. In other embodiments, 30ml bone marrow aspirate is obtained from a patient. In other embodiments, 40ml bone marrow aspirate is obtained from a patient. In other embodiments, 50ml bone marrow aspirate is obtained from a patient.

In some embodiments of the invention, the number of PBMC generated from about 10-50ml bone marrow aspirate is about 5X 10 ⁷ to about 10X 10 ⁷ PBMC. In other embodiments, the number of PMBC produced is about 7 x 10 ⁷ PBMCs.

In some embodiments of the invention, about 5×10 ⁷ to about 10×10 ⁷ PBMCs produce about 0.5×10 ⁶ to about 1.5×10 ⁶ expansion starting cell material. In some embodiments of the invention, about 1×10 ⁶ expansion-starting cell materials are produced.

In some embodiments of the invention, the total number of MILs collected at the end of the amplification cycle is about 0.01x10 ⁹ to about 1 x 10 ⁹, about 0.05x10 ⁹ to about 0.9x10 ⁹, about 0.1x10 ⁹ to about 0.85 x 10 ⁹, about 0.15x10 ⁹ to about 0.7 x 10 ⁹, about 0.2x10 ⁹ to about 0.65 x 10 ⁹, about 0.25x10 ⁹ to about 0.6x10 ⁹, about 0.3x10 ⁹ to about 0.55 x 10 ⁹, about 0.35x10 ⁹ to about 0.5x10 ⁹, or about 0.4x10 ⁹ to about 0.45 x 10 ⁹.

In some embodiments of the invention, 12×10 ⁶ PBMCs derived from bone marrow aspirate produce about 1.4×10 ⁵ starting cell materials, which at the end of the expansion process produce about 1.1×10 ⁷ MILs.

In some embodiments of the invention, MILs amplified from bone marrow PBMCs using MILs method 3 described above comprise a higher proportion of cd8+ cells and a fewer number of lag3+ and pd1+ cells than MILs amplified using MILs method 1 or MILs method 2. In some embodiments of the invention, PBLs amplified from blood PBMCs using MILs method 3 described above comprise a higher proportion of cd8+ cells and increased ifnγ production compared to PBLs amplified using MILs method 1 or MILs method 2.

In some embodiments of the invention, the clinical dose of MILs suitable for Acute Myelogenous Leukemia (AML) patients is in the range of about 4 x 10 ⁸ to about 2.5 x 10 ⁹ MILs. In other embodiments, the number of MILs provided in the pharmaceutical composition of the invention is 9.5 x 10 ⁸ MILs. In other embodiments, the number of MILs provided in the pharmaceutical composition of the invention is 4.1 x 10 ⁸. In other embodiments, the number of MILs provided in the pharmaceutical composition of the invention is 2.2x10 ⁹.

In any of the foregoing embodiments, the PBMCs may be derived from whole blood samples, bone marrow, obtained by apheresis, derived from buffy coat, or obtained from any other method known in the art for obtaining PBMCs.

Gen 2TIL manufacturing Process

An exemplary set of TIL procedures, known as Gen 2 (also known as process 2A), is depicted in fig. 1 and 2, which contains some of these features. An example of Gen 2 is shown in fig. 2.

As described herein, the present invention may include steps associated with re-stimulating cryopreserved TILs to increase their metabolic activity and thus relative health prior to implantation into a patient, and methods of testing such metabolic health. As generally summarized herein, TILs are typically obtained from patient samples and are manipulated to amplify their number prior to transplantation into a patient. In some embodiments, the TIL is optionally genetically manipulated, as described below.

In some embodiments, the TIL may be cryopreserved. After thawing, it may also be re-stimulated to increase its metabolism prior to infusion into a patient.

In some embodiments, the first amplification (including the process called pre-REP and the process shown in fig. 1 as step a) is shortened to 3 to 14 days, and the second amplification (including the process called REP and the process shown in fig. 1 as step B) is shortened to 7 to 14 days, as discussed in detail below and in the examples and illustrations. In some embodiments, the first amplification (e.g., the amplification described in step B of fig. 1) is shortened to 11 days and the second amplification (e.g., the amplification described in step D of fig. 1) is shortened to 11 days. In some embodiments, the combination of the first amplification and the second amplification (e.g., the amplification described in fig. 1 as step B and step D) is shortened to 22 days, as discussed in detail below and in the examples and illustrations.

The "step" designation A, B, C, et al, hereinafter refers to fig. 1 and to certain embodiments described herein. The order of steps in the following and in fig. 1 is exemplary, and the application and methods disclosed herein encompass any combination or order of steps, as well as additional steps, repetition of steps, and/or omission of steps.

A. Step A: obtaining a patient tumor sample

Generally, TIL is initially obtained from a patient tumor sample, and subsequently expanded into a larger population for further manipulation as described herein, optionally restimulation as outlined herein, cryopreservation, and optionally evaluation of phenotypic and metabolic parameters that are indicators of TIL health.

Patient tumor samples may be obtained using methods known in the art, typically by surgical excision, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining samples containing a mixture of tumor and TIL cells. In some embodiments, multi-lesion sampling is used. In some embodiments, surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells include multifocal sampling (i.e., obtaining samples from more than one tumor site and/or location in a patient and at the same location or immediately adjacent more than one tumor). In general, a tumor sample may be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematopoietic malignancy. The solid tumor may be lung tissue. In some embodiments, suitable TILs are obtained from non-small cell lung cancer (NSCLC). The solid tumor may be a skin tissue. In some embodiments, a suitable TIL is obtained from melanoma.

Once obtained, the tumor sample is typically broken into small pieces of between 1mm ³ and about 8mm ³, with about 2 to 3mm ³ being particularly suitable, using sharp instrument splitting. In some embodiments, TIL is cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by culturing in an enzyme medium (e.g., roscoe park cancer institute (Roswell Park Memorial Institute; RPMI) 1640 buffer, 2mM glutamate, 10mcg/mL gentamicin (gentamicine), 30 units/mL DNase, and 1.0mg/mL collagenase), followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by: tumors were placed in enzyme medium, mechanically dissociated for about 1 min, followed by incubation in 5% CO ₂ at 37 ℃ for 30 min, followed by repeated mechanical dissociation and incubation cycles under the aforementioned conditions until only small tissue pieces were present. At the end of this process, if the cell suspension contains a large number of erythrocytes or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. patent application publication 2012/0244233 A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in the methods of amplifying TIL or methods of treating cancer in any of the embodiments described herein.

The tumor dissociating enzyme mixture may comprise one or more dissociating (digestive) enzymes, such as, but not limited to, collagenase (including any mixed or type of collagenase), accutase ^TM、Accumax^TM, hyaluronidase (hyaluronidase), neutral protease (dispase), chymotrypsin (chymotrypsin), chymopapain (chymopapain), trypsin (trypsin), caseinase (caseinase), elastase (elastase), papain (papain), XIV-type protease (pronase (pronase)), deoxyribonuclease I (dnase), trypsin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof.

In some embodiments, the dissociating enzyme is reconstituted by a freeze-drying enzyme (reconstitute). In some embodiments, the lyophilized enzyme is reconstituted in a quantity of sterile buffer (e.g., HBSS).

In some cases, the collagenase (e.g., no animal-derived collagenase type 1) is reconstituted in 10mL of sterile HBSS or another buffer. The concentration of lyophilized stock enzyme may be 2892PZ U per vial. In some embodiments, the collagenase is reconstituted in 5mL to 15mL buffer. In some embodiments, the collagenase stock solution ranges from about 100PZ U/mL to about 400PZ U/mL after reconstitution, e.g., from about 100PZ U/mL to about 400PZ U/mL, from about 100PZ U/mL to about 350PZ U/mL, from about 100PZ U/mL to about 300PZ U/mL, from about 150PZ U/mL to about 400PZ U/mL, from about 100PZ U/mL, about 150PZ U/mL, about 200PZ U/mL, about 210PZ U/mL, about 220PZ U/mL, about 230 PZ U/mL, about 240PZU/mL, about 250PZ U/mL, about 260PZ U/mL, about 270PZ U/mL, about 280PZ U/mL, about 289.2PZ U/mL, about 300PZ U/mL, about 350PZ U/mL, or about 400PZ U/mL.

In some embodiments, the neutral protease is reconstituted in 1mL of sterile HBSS or another buffer. The concentration of lyophilized stock enzyme may be 175DMC U per vial. In some embodiments, after reconstitution, the neutral protease stock solution ranges from about 100DMC/mL to about 400DMC/mL, e.g., from about 100DMC/mL to about 400DMC/mL, from about 100DMC/mL to about 350DMC/mL, from about 100DMC/mL to about 300DMC/mL, from about 150DMC/mL to about 400DMC/mL, from about 100DMC/mL, about 110DMC/mL, about 120DMC/mL, about 130DMC/mL, about 140DMC/mL, about 150DMC/mL, about 160DMC/mL, about 170DMC/mL, about 175DMC/mL, about 180DMC/mL, about 190DMC/mL, about 200DMC/mL, about 250DMC/mL, about 300DMC/mL, about 350DMC/mL, or about 400DMC/mL.

In some embodiments, dnase I is reconstituted in 1mL sterile HBSS or another buffer. The concentration of lyophilized stock enzyme was 4KU per vial. In some embodiments, the DNase I stock solution after reconstitution ranges from about 1KU/mL to 10KU/mL, for example, about 1KU/mL, about 2KU/mL, about 3KU/mL, about 4KU/mL, about 5KU/mL, about 6KU/mL, about 7KU/mL, about 8KU/mL, about 9KU/mL, or about 10KU/mL.

In some embodiments, the stock solution of enzyme is variable and it may be desirable to determine the concentration. In some embodiments, the concentration of the lyophilized stock solution can be checked. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture comprises about 10.2. Mu.l neutral protease (0.36 DMC/mL), 21.3. Mu.l collagenase (1.2 PZ/mL) and 250. Mu.l DNase I (200U/mL) in about 4.7mL sterile HBSS.

As noted above, in some embodiments, the TIL is derived from a solid tumor. In some embodiments, the solid tumor is not disrupted. In some embodiments, the solid tumor is not disrupted and the enzyme digestion is performed with the whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase at 37 ℃ for 1 to 2 hours at 5% CO ₂. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase at 37 ℃ with 5% CO ₂ under rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37 ℃, 5% CO ₂, constant rotation. In some embodiments, the intact tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with the lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is 100mg/mL of 10X working stock.

In some embodiments, the enzyme mixture comprises dnase. In some embodiments, the working stock of dnase is 10,000iu/mL of 10X working stock.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is 10mg/mL of 10X working stock.

In some embodiments, the enzyme mixture comprises 10mg/mL collagenase, 1000IU/mL DNase, and 1mg/mL hyaluronidase.

In some embodiments, the enzyme mixture comprises 10mg/mL collagenase, 500IU/mL DNase, and 1mg/mL hyaluronidase.

In general, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, crushing includes physical crushing, including, for example, segmentation and digestion. In some embodiments, the crushing is physical crushing. In some embodiments, the fracture is split. In some embodiments, the disruption is by digestion. In some embodiments, the TIL may be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or disrupting tumor samples obtained from the patient.

In some embodiments, when the tumor is a solid tumor, the tumor undergoes physical disruption after obtaining a tumor sample, e.g., in step a (as provided in fig. 1). In some embodiments, the crushing occurs prior to cryopreservation. In some embodiments, the crushing occurs after cryopreservation. In some embodiments, the disruption is performed after the tumor is obtained and without any cryopreservation. In some embodiments, the tumor is disrupted and more than 10, 20, 30, 40 fragments or pieces are placed in each container for a first amplification. In some embodiments, the tumor is disrupted and 30 or 40 fragments or fragments are placed in each container for first expansion. In some embodiments, the tumor is disrupted and 40 fragments or fragments are placed in each container for first expansion. In some embodiments, the plurality of fragments comprises from about 4 to about 50 fragments, each fragment having a volume of about 27mm ³. In some embodiments, the plurality of fragments comprises from about 30 to about 60 fragments, with a total volume of from about 1300mm ³ to about 1500mm ³. In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350mm ³. In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the TIL is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp segmentation. In some embodiments, the tumor fragments are between about 1mm ³ and 10mm ³. In some embodiments, the tumor fragments are between about 1mm ³ and 8mm ³. In some embodiments, the tumor fragments are about 1mm ³. In some embodiments, the tumor fragments are about 2mm ³. In some embodiments, the tumor fragments are about 3mm ³. In some embodiments, the tumor fragments are about 4mm ³. In some embodiments, the tumor fragments are about 5mm ³. In some embodiments, the tumor fragments are about 6mm ³. In some embodiments, the tumor fragments are about 7mm ³. In some embodiments, the tumor fragments are about 8mm ³. In some embodiments, the tumor fragments are about 9mm ³. In some embodiments, the tumor fragments are about 10mm ³. In some embodiments, the tumor is 1-4mm. In some embodiments, the tumor is 1mm×1mm. In some embodiments, the tumor is 2mm x 2mm. In some embodiments, the tumor is 3mm×3mm. In some embodiments, the tumor is 4mm by 4mm.

In some embodiments, the tumor is resected to minimize the amount of hemorrhagic, necrotic, and/or adipose tissue on each mass. In some embodiments, the tumor is resected to minimize the amount of hemorrhagic tissue on each mass. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each mass. In some embodiments, the tumor is resected to minimize the amount of adipose tissue on each mass.

In some embodiments, tumor disruption is performed to maintain tumor internal structure. In some embodiments, tumor disruption is performed without a sawing action using a scalpel. In some embodiments, the TIL is obtained from tumor digests. In some embodiments, the tumor digests are produced by incubation in an enzyme medium (such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10mg/mL gentamicin, 30U/mL dnase, and 1.0mg/mL collagenase), followed by mechanical dissociation (GENTLEMACS of the biotechnology of obumeday, california). After placing the tumor in the enzyme medium, the tumor may be dissociated mechanically for about 1 minute. The solution may then be incubated in 5% CO ₂ at 37℃for 30 minutes and then again mechanically disrupted for about 1 minute. After an additional 30 minutes incubation in 5% CO ₂ at 37℃the tumor can be mechanically destroyed a third time for about 1 minute. In some embodiments, if bulk tissue is still present after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, whether or not incubated in 5% CO ₂ for 30 minutes at 37 ℃. In some embodiments, at the end of the final culture, if the cell suspension contains a large number of erythrocytes or dead cells, density gradient separation can be performed using Ficoll to remove these cells.

In some embodiments, the cell suspension collected prior to the first expansion step is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, the cells may optionally be frozen after sample collection and stored frozen prior to entering the expansion described in step B (which is described in further detail below and illustrated in fig. 1 and 8).

1. Pleural effusion T cells and TIL

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion according to the processes described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of T cells or TILs for expansion according to the processes described herein is a sample derived from pleural effusion. See, for example, the method described in U.S. patent publication No. US2014/0295426, which is incorporated by reference herein in its entirety for all purposes.

In some embodiments, any pleural or pleural effusion suspected of and/or containing TIL may be employed. Such samples may be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample may be derived from secondary metastatic cancer cells derived from another organ (e.g., breast, ovary, colon, or prostate). In some embodiments, the sample used in the amplification methods described herein is pleural effusion (pleural exudate). In some embodiments, the sample used in the amplification methods described herein is pleural effusion (pleural transudate). Other biological samples may include other slurries containing TIL, including, for example, ascites fluid from the abdomen or pancreatic cyst fluid. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and the lung have mesothelial cell lines and fluid forms in the thorax and abdominal cavities in the same malignant event, in some embodiments such fluids contain TIL. In some embodiments where the disclosed methods utilize pleural fluid, the same methods may be performed using ascites or other cyst fluid containing TIL to achieve similar results.

In some embodiments, pleural fluid is removed directly from the patient in untreated form. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube (e.g., EDTA or heparin tube) prior to the further treatment step. In some embodiments, the untreated pleural fluid is placed on a standard prior to the further treatment stepIn a tube (Veridex). In some embodiments, the sample is placed in the CellSave tube immediately after collection from the patient to avoid a decrease in the number of active TILs. If left in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4 ℃. In some embodiments, the sample is placed in a suitable collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in a suitable collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4 ℃.

In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is a 1:10 pleural fluid to diluent. In other embodiments, the dilution is a 1:9 pleural fluid to diluent. In other embodiments, the dilution is a 1:8 pleural fluid to diluent. In other embodiments, the dilution is a 1:5 pleural fluid to diluent. In other embodiments, the dilution is a 1:2 pleural fluid to diluent. In other embodiments, the dilution is a 1:1 pleural fluid to diluent. In some embodiments, the diluent comprises saline, phosphate buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, samples are placed in CellSave tubes immediately after collection and dilution from the patient to avoid a decrease in active TIL, which if left in untreated pleural fluid, may decrease significantly over 24 to 48 hours, even at 4 ℃. In some embodiments, the pleural fluid sample is placed in a suitable collection tube 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal and dilution from the patient. In some embodiments, the pleural fluid sample is placed in a suitable collection tube 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient and dilution at 4 ℃.

In still other embodiments, the pleural fluid sample is concentrated by conventional means prior to the further processing step. In some embodiments, pre-treatment of pleural fluid is preferred in cases where pleural fluid must be cryopreserved for shipment to a laboratory where the method is performed or for subsequent analysis (e.g., 24 to 48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is removed from the subject and re-suspending the centrifugate or precipitate in buffer. In some embodiments, the pleural fluid sample is centrifuged and resuspended multiple times and then cryopreserved for shipment or later analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated by using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in the further treatment is prepared by filtering the fluid through a filter containing a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the pores in the membrane may be at least 4 μm in diameter. In other embodiments, the pore size may be above 5 μΜ, and in other embodiments, may be any of 6 μΜ,7 μΜ,8 μΜ, 9 μΜ or 10 μΜ. After filtration, the cells (including TIL) retained by the membrane may be washed from the membrane into a suitable physiologically acceptable buffer. The cells concentrated in this way (including TIL) can then be used in further processing steps of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysing agent that differentially lyses the non-nucleated red blood cells present in the sample. In some embodiments, where the pleural fluid contains a large number of RBCs, this step is performed prior to further processing steps. Suitable lysing reagents include a single lysing reagent or a lysing reagent and a quenching reagent, or a lysing reagent, a quenching reagent, and an immobilization reagent. Suitable lysis systems are commercially available, including the BD PHARM LYSE ^TM system (Bidi medical Co., becton Dickenson). Other lysis systems include VERSALYSE ^TM systems, FACSlyse ^TM systems (Bidi medical Co.), immunoprep ^TM systems or Erythrolyse II systems (Beckman Coulter, inc.) or ammonium chloride systems. In some embodiments, the lysing agent may vary with the primary requirements, which are efficient lysis of erythrocytes and conservation of TIL and phenotypic characteristics of TIL in pleural fluid. In addition to using a single reagent for cleavage, a cleavage system suitable for use in the methods described herein may include a second reagent, such as a Stabilyse ^TM reagent (Beckmann Coulter) that quenches or delays the action of the cleavage reagent during the remaining steps of the method. Depending on the choice of cleavage reagent or the preferred implementation of the method, conventional immobilization reagents may also be used.

In some embodiments, untreated, diluted, or multi-centrifugation or treated pleural fluid samples as described above are cryopreserved at a temperature of about-140 ℃ followed by further processing and/or amplification as provided herein.

B. And (B) step (B): first amplification

In some embodiments, the methods of the present invention provide for obtaining a young TIL that is capable of achieving an increased replication cycle upon administration to a subject/patient and thus may provide additional therapeutic benefits compared to an older TIL (i.e., a TIL that further undergoes more rounds of replication prior to administration to the subject/patient). The characteristics of young TILs have been described in the literature, for example Donia et al, journal of scandinavia immunology (scand.j.immunol.) 2012,75,157-167; dudley et al, clinical cancer research 2010,16,6122-6131; huang et al, journal of immunology 2005,28,258-267; besser et al, clinical cancer research 2013,19, OF1-OF9; besser et al, journal of immunology 2009,32:415-423; robbins et al, journal of immunology 2004,173,7125-7130; shen et al, journal of immunology 2007,30,123-129; zhou et al, journal of immunology 2005,28,53-62; and Tran et al, journal of immunology 2008,31,742-751, each of which is incorporated herein by reference.

The various antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene segments: v (variable region), D (variable region), J (junction region) and C (constant region) determine the binding specificity and downstream application of immunoglobulins to T Cell Receptors (TCRs). The present invention provides a method for producing TIL that exhibits and increases T cell reservoir diversity. In some embodiments, the TIL obtained by the methods of the invention exhibits increased T cell reservoir diversity. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell reservoir diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in fig. 1. In some embodiments, the TIL obtained by the methods of the invention exhibits increased T cell reservoir diversity compared to freshly collected TIL and/or TIL prepared using the methods referred to as process 1C as exemplified in fig. 5 and/or 6. In some embodiments, the TIL obtained in the first expansion exhibits increased T cell reservoir diversity. In some embodiments, increasing the diversity is increasing the diversity of immunoglobulins and/or T cell receptor diversity. In some embodiments, the diversity is present in the immunoglobulin and in the heavy chain of the immunoglobulin. In some embodiments, the diversity is present in the immunoglobulin and in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T Cell Receptor (TCR) α and/or β is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, TCRab (i.e., tcra/β) is expressed in increased.

Following dissection or digestion of tumor fragments, e.g., as described in step a of fig. 1, the resulting cells are cultured in serum containing IL-2 under conditions conducive to TIL growth relative to the tumor and other cells. In some embodiments, tumor digests are incubated in 2mL wells in medium comprising inactivated human AB serum with 6000IU/mL IL-2. This primary cell population is cultured for a period of days, typically 3 to 14 days, resulting in a population of bulk TIL of typically about 1 x 10 ⁸ bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, resulting in a subject TIL population of typically about 1 x 10 ⁸ subject TIL cells. In some embodiments, this primary cell population is cultured for a period of 10 to 14 days, resulting in a subject TIL population of typically about 1 x 10 ⁸ subject TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a host TIL population of typically about 1 x 10 ⁸ host TIL cells.

In some embodiments, amplification of the TIL may be performed using an initial subject TIL amplification step (e.g., those described in step B of fig. 1, which may include a process called pre-REP) as described below and herein, followed by a second amplification (step D, including a process called rapid amplification protocol (REP) step) as described below and herein, followed by an optional cryopreservation, followed by a second step D (including a process called re-stimulus REP step) as described below and herein. TIL obtained from this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

In embodiments where TIL culture is initiated in 24 well plates, for example, using a Costar 24 well cell culture cluster, flat bottom (Corning Incorporated, corning, NY, each well can be inoculated with 1 x 10 ⁶ tumor digest cells or one tumor fragment in 2mL of Complete Medium (CM) with IL-2 (6000 IU/mL; chiron corp., emeryville, CA.) in some embodiments, the tumor fragments are between about 1mm ³ and 10mm ³.

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for medium). In some embodiments, the CM of step B consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25mM Hepes, and 10mg/mL gentamicin. In an embodiment of initiation of culture in a gas-permeable flask (e.g., G-REX-10;Wilson Wolf Manufacturing,New Brighton,MN) having a capacity of 40mL and a 10CM ² gas-permeable silicon bottom, each flask is loaded with 10-40mL CM containing 10-40X 10 ⁶ live tumor digest cells or 5 to 30 tumor fragments and IL-2. G-REX-10 and 24 well plates were each incubated in a wet gas incubator at 37℃at 5% CO ₂, half of the medium was removed 5 days after initiation of incubation, replaced with fresh CM and IL-2, and half of the medium was replaced every 2 to 3 days after 5 days.

In some embodiments, the medium used in the amplification process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement. In some embodiments, serum-free or defined media is used to prevent and/or reduce experimental variation due in part to batch-to-batch variation of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement. In some embodiments, the basal cell culture Medium includes, but is not limited to, CTS ^TMOpTmizer^TM T cell expansion basal Medium, CTS ^TMOpTmizer^TM T cell expansion SFM, CTS ^TM AIM-V Medium, CTS ^TMAIM-V SFM、LymphoONE^TM T cell expansion no Xeno Medium, darby's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), isagl Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential Medium (αMEM), grasgang minimal essential Medium (G-MEM), RPMI growth Medium, and Iskov modified Darby's Medium (Iscove's Modified Dulbecco's Medium).

In some embodiments, the serum supplement or serum replacement includes (but is not limited to) more than one of the following: CTS ^TM OpTmizer T cell-expanded serum supplement, CTS ^TM immune cell serum replacement, one or more albumin or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and compounds containing trace element moieties Ag⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺ and Zr ⁴⁺. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS ^TMOpTmizer^TM T cell immune cell serum replacement is used with conventional growth media including, but not limited to, CTS ^TMOpTmizer^TM T cell expansion basal media, CTS ^TMOpTmizer^TM T cell expansion SFM, CTS ^TM AIM-V media, CST ^TMAIM-V SFM、LymphoONE^TM T cell expansion no Xeno media, darburg Modified Eagle's Medium (DMEM), minimal Essential Media (MEM), eagle Basal Media (BME), RPMI 1640, F-10, F-12, minimal essential media (αmem), glasgang minimal essential media (G-MEM), RPMI growth media, and iskov modified darburg media.

In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% based on the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS ^TMOpTmizer^TM T cell expansion SFM (sameir feishier technology). Any CTS ^TMOpTmizer^TM formulation may be used in the present invention. CTS ^TMOpTmizer^TM T cell expansion SFM is a combination of 1L CTS ^TMOpTmizer^TM T cell expansion basal medium and 26mL CTS ^TMOpTmizer^TM T cell expansion supplement mixed together prior to use. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sameir feishier technology). In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), the final concentration of 2-mercaptoethanol in the medium is 55 μm.

In some embodiments, the defined medium is CTS ^TMOpTmizer^TM T cell expansion SFM (sammer feishier technology). Any CTS ^TMOpTmizer^TM formulation may be used in the present invention. CTS ^TMOpTmizer^TM T cell expansion SFM is a combination of 1L CTS ^TMOpTmizer^TM T cell expansion basal medium and 26mL CTS ^TMOpTmizer^TM T cell expansion supplement mixed together prior to use. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), with a final concentration of 55 μm in the medium of 2-mercaptoethanol.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine at a concentration of about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5mM (i.e.,). In some embodiments, the serum-free medium or defined medium is supplemented with glutamine at a concentration of about 2mM (i.e./>))。

In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55. Mu.M.

In some embodiments, defined media as described in International PCT publication No. WO/1998/030679, incorporated herein by reference, may be used in the present invention. In this disclosure, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more components selected from the group consisting of: more than one albumin or albumin substitute, more than one amino acid, more than one vitamin, more than one transferrin or transferrin substitute, more than one antioxidant, more than one insulin or insulin substitute, more than one collagen precursor, more than one trace element, and more than one antibiotic. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β -mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: more than one amino acid, more than one vitamin, more than one transferrin or transferrin substitute, more than one antioxidant, more than one insulin or insulin substitute, more than one collagen precursor, and more than one trace element. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and compounds containing trace element moieties Ag⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺ and Zr ⁴⁺. In some embodiments, the basal cell culture medium is selected from the group consisting of: darburg's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (αmem), grange minimal essential medium (G-MEM), RPMI growth medium, and eoskov modified darburg's medium.

In some embodiments, the concentration of glycine in the ingredient medium is determined to be in the range of about 5 to 200mg/L, the concentration of L-histidine is about 5 to 250mg/L, the concentration of L-isoleucine is about 5 to 300mg/L, the concentration of L-methionine is about 5 to 200mg/L, the concentration of L-phenylalanine is about 5 to 400mg/L, the concentration of L-proline is about 1 to 1000mg/L, the concentration of L-hydroxyproline is about 1 to 45mg/L, the concentration of L-serine is about 1 to 250mg/L, the concentration of L-threonine is about 10 to 500mg/L, the concentration of L-tryptophan is about 2 to 110mg/L, the concentration of L-tyrosine is about 3 to 175mg/L, the concentration of L-valine is about 5 to 500mg/L, the concentration of thiamine is about 1 to 20mg/L, the concentration of reduced glutathione is about 1 to 20mg/L, the concentration of L-ascorbic acid-2-phosphate is about 1 to 200mg/L, the concentration of iron-saturated protein is about 1 to 50mg/L, the concentration of sodium for example, the concentration of insulin is about 0.0001 to 0.01 mg/LI) Is about 5000 to 50,000mg/L.

In some embodiments, the non-trace element fraction ingredients in the defined ingredient medium are present in the concentration ranges listed in the column entitled "concentration ranges in 1X medium" in table 12 below. In other embodiments, the non-trace element fraction ingredients in the defined ingredient medium are present at the final concentrations listed in the column entitled "preferred embodiment of Medium" 1X Medium in Table 12. In other embodiments, the defined medium is a basal cell medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace amounts of ingredients at concentrations listed in the type and heading "preferred embodiments of supplement" column in table 12 below.

Table 12: concentration of non-trace element fraction

In some embodiments, the defined medium has an osmolality between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7g/L or about 2.2g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100. Mu.M), 2-mercaptoethanol (final concentration of about 100. Mu.M).

In some embodiments, the defined medium described in Smith et al, clinical and transformation Immunology (CLIN TRANSL Immunology) 4 (1) 2015 (doi: 10.1038/cti.2014.31) is suitable for use in the present invention. Briefly, RPMI or CTS ^TMOpTmizer^TM was used as basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS ^TM immune cell serum replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable containers is unfiltered. The use of unfiltered cell culture media can simplify the procedure required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second gas permeable containers lacks beta-mercaptoethanol (BME or beta ME; also known as 2-mercaptoethanol, CAS 60-24-2).

After preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions conducive to TIL growth relative to the tumor and other cells. In some embodiments, tumor digests are incubated in 2mL wells in medium comprising inactivated human AB serum (or in some cases, in the presence of a population of APC cells, as described herein) and 6000IU/mL IL-2. This primary cell population is cultured for a period of days, typically 10 to 14 days, resulting in a population of bulk TIL of typically about 1 x 10 ⁸ bulk TIL cells. In some embodiments, the growth medium comprises IL-2 or a variant thereof during the first amplification. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1mg vial of IL-2 stock solution has a specific activity of 20 to 30X 10 ⁶ IU/mg. In some embodiments, a 1mg vial of IL-2 stock has a specific activity of 20X 10 ⁶ IU/mg. In some embodiments, a 1mg vial of IL-2 stock has a specific activity of 25X 10 ⁶ IU/mg. In some embodiments, a 1mg vial of IL-2 stock has a specific activity of 30X 10 ⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4 to 8X 10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5 to 7X 10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6X 10 ⁶ IU/mg IL-2. In some embodiments, IL-2 stock solutions are prepared as described in example 5. In some embodiments, the first amplification medium comprises about 10,000IU/mL IL-2, about 9,000IU/mL IL-2, about 8,000IU/mL IL-2, about 7,000IU/mL IL-2, about 6000IU/mL IL-2, or about 5,000IU/mL IL-2. In some embodiments, the first amplification medium comprises about 9,000IU/mL IL-2 to about 5,000IU/mL IL-2. In some embodiments, the first amplification medium comprises about 8,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the first amplification medium comprises about 7,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the first amplification medium comprises about 6,000IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000IU/mL, about 1500IU/mL, about 2000IU/mL, about 2500IU/mL, about 3000IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, or about 8000IU/mL IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000IU/mL, between 2000 and 3000IU/mL, between 3000 and 4000IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000IU/mL, between 6000 and 7000IU/mL, between 7000 and 8000IU/mL, or about 8000IU/mL of IL-2.

In some embodiments, the first amplification medium comprises about 500IU/mL IL-15, about 400IU/mL IL-15, about 300IU/mL IL-15, about 200IU/mL IL-15, about 180IU/mL IL-15, about 160IU/mL IL-15, about 140IU/mL IL-15, about 120IU/mL IL-15, or about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 500IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 400IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 300IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 200IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the first amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mLIL-21. In some embodiments, the first amplification medium comprises about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the first amplification medium comprises about 2IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21.

In some embodiments, the cell culture medium comprises an anti-CD 3 agonist antibody, such as an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1. Mu.g/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises OKT-3 antibodies between 0.1ng/mL and 1ng/mL, between 1ng/mL and 5ng/mL, between 5ng/mL and 10ng/mL, between 10ng/mL and 20ng/mL, between 20ng/mL and 30ng/mL, between 30ng/mL and 40ng/mL, between 40ng/mL and 50ng/mL, and between 50ng/mL and 100 ng/mL. In some embodiments, the cell culture medium does not comprise an OKT-3 antibody. In some embodiments, the OKT-3 antibody is Moromolizumab. See, e.g., table 1.

In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: wu Ruilu mab, wu Tumu mab, EU-101, fusion proteins, fragments, derivatives, variants, biological analogs, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 μg/mL and 100 μg/mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 μg/mL and 40 μg/mL.

In some embodiments, the cell culture medium further comprises IL-2 at an initial concentration of about 3000IU/mL and OKT-3 antibody at an initial concentration of about 30ng/mL in addition to the one or more TNFRSF agonists, wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for medium). In some embodiments, it is referred to as CM1 (medium 1). In some embodiments, CM consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25mM Hepes, and 10mg/mL gentamicin. In an embodiment where culture is initiated in a gas-permeable flask (e.g., G-REX10; wilson Wolf Manufacturing, new Brighton, MN) having a capacity of 40mL and a 10CM ² gas-permeable silicon bottom, each flask is loaded with 10 to 40mL CM with IL-2 containing 10 to 40X 10 ⁶ live tumor digest cells or 5 to 30 tumor fragments. Both G-REX10 and 24 well plates were incubated in a wet gas incubator at 37℃at 5% CO ₂, half of the medium was removed 5 days after the start of incubation, replaced with fresh CM and IL-2, and half of the medium was replaced every 2 to 3 days after the 5 th day. In some embodiments, CM is CM1 described in the examples, see example 1. In some embodiments, the first expansion occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium comprises IL-2.

In some embodiments, the first amplification (including processes such as those described in step B of fig. 1, which may include those sometimes referred to as pre-REP) is shortened to 3 to 14 days, as described in the examples and illustrations. In some embodiments, the first amplification (including, for example, the nx process described in step B of fig. 1, which may include those processes sometimes referred to as pre-REP) is shortened to 7 to 14 days, as described in the examples and shown in fig. 4 and 5, and includes, for example, the amplification described in step B of fig. 1. In some embodiments, the first amplification of step B is shortened to 10 to 14 days. In some embodiments, the first amplification is shortened to 11 days, e.g., as described in the amplification described in step B of fig. 1.

In some embodiments, the first TIL amplification may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL amplification may be performed for 1 day to 14 days. In some embodiments, the first TIL amplification may be performed for 2 days to 14 days. In some embodiments, the first TIL amplification may be performed for 3 days to 14 days. In some embodiments, the first TIL amplification may be performed for 4 days to 14 days. In some embodiments, the first TIL amplification may be performed for 5 days to 14 days. In some embodiments, the first TIL amplification may be performed for 6 days to 14 days. In some embodiments, the first TIL amplification may be performed for 7 days to 14 days. In some embodiments, the first TIL amplification may be performed for 8 days to 14 days. In some embodiments, the first TIL amplification may be performed for 9 days to 14 days. In some embodiments, the first TIL amplification may be performed for 10 days to 14 days. In some embodiments, the first TIL amplification may be performed for 11 days to 14 days. In some embodiments, the first TIL amplification may be performed for 12 days to 14 days. In some embodiments, the first TIL amplification may be performed for 13 days to 14 days. In some embodiments, the first TIL amplification may be performed for 14 days. In some embodiments, the first TIL amplification may be performed for 1 day to 11 days. In some embodiments, the first TIL amplification may be performed for 2 days to 11 days. In some embodiments, the first TIL amplification may be performed for 3 days to 11 days. In some embodiments, the first TIL amplification may be performed for 4 days to 11 days. In some embodiments, the first TIL amplification may be performed for 5 days to 11 days. In some embodiments, the first TIL amplification may be performed for 6 days to 11 days. In some embodiments, the first TIL amplification may be performed for 7 days to 11 days. In some embodiments, the first TIL amplification may be performed for 8 days to 11 days. In some embodiments, the first TIL amplification may be performed for 9 days to 11 days. In some embodiments, the first TIL amplification may be performed for 10 days to 11 days. In some embodiments, the first TIL amplification may be performed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is used as the combination during the first amplification. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included during the first amplification, including, for example, during the step B process according to FIG. 1 and described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is used as the combination during the first amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the step B process according to FIG. 1 and as described herein.

In some embodiments, as described in the examples and illustrations, the first amplification (including a process known as prerep; e.g., according to step B) of fig. 1) process is shortened to 3 to 14 days. In some embodiments, the first amplification of step B is shortened to 7 to 14 days. In some embodiments, the first amplification of step B is shortened to 10 to 14 days. In some embodiments, the first amplification is shortened to 11 days.

In some embodiments, the first amplification is performed in a closed system bioreactor, e.g., according to step B of fig. 1. In some embodiments, the TIL amplification as described herein is performed using a closed system. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

1. Cytokines and other additives

The amplification methods described herein generally use media with high doses of cytokines (particularly IL-2), as known in the art.

Or combinations of cytokines for rapid amplification and/or second amplification of TIL are also possible, as described in U.S. patent application publication No. US2017/0107490 A1, using two or more of the combinations of IL-2, IL-15 and IL-21, the disclosures of which are incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, the latter having particular utility in many embodiments. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, in particular T cells as described therein.

In some embodiments, step B may also comprise adding OKT-3 antibody or moromiab to the medium, as described elsewhere herein. In some embodiments, step B may also include adding a 4-1BB agonist to the medium, as described elsewhere herein. In some embodiments, step B may also comprise adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator activated receptor gamma coactivator I-alpha agonists, including proliferator activated receptor (PPAR) -gamma agonists, such as thiazolidinedione compounds, may be used in the medium during step B, as described in U.S. patent application publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. step C: transition from first to second amplification

In some cases, a population of bulk TILs obtained from the first amplification, including, for example, a population of TILs obtained from step B, e.g., as shown in fig. 1, may be immediately cryopreserved using the protocol described below. Or the TIL population obtained from the first amplification (referred to as the second TIL population) may undergo a second amplification (which may include an amplification sometimes referred to as REP) and then be cryopreserved as described below. Similarly, where a genetically modified TIL is to be used in therapy, a first TIL population (sometimes referred to as a subject TIL population) or a second TIL population (which in some embodiments may include a population referred to as a REP TIL population) may be genetically modified for appropriate treatment prior to amplification or after the first amplification and prior to the second amplification.

In some embodiments, TIL obtained from the first amplification (e.g., from step B as indicated in fig. 1) is stored until a phenotypic analysis for selection is performed. In some embodiments, TIL obtained from the first amplification (e.g., from step B as indicated in fig. 1) is not stored and proceeds directly to the second amplification. In some embodiments, TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from first amplification to second amplification is performed about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after disruption. In some embodiments, the transition from the first amplification to the second amplification is performed about 3 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed about 4 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification occurs from about 4 days to about 10 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification is performed about 7 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification occurs about 14 days after disruption.

In some embodiments, the transition from first amplification to second amplification is performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after disruption. In some embodiments, the transition from the first amplification to the second amplification is performed 1 day to 14 days after the disruption is performed. In some embodiments, the first TIL amplification may be performed for 2 days to 14 days. In some embodiments, the transition from the first amplification to the second amplification is performed 3 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 4 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 5 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed between 6 days and 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 7 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed between 8 days and 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 9 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed between 10 days and 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 11 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 12 days to 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed between 13 days and 14 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 14 days after the disruption. In some embodiments, the transition from the first amplification to the second amplification is performed 1 day to 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed from 2 days to 11 days after the disruption. In some embodiments, the transition from the first amplification to the second amplification is performed 3 days to 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 4 days to 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 5 days to 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed between 6 days and 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 7 days to 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed between 8 days and 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 9 days to 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed between 10 days and 11 days after the disruption is performed. In some embodiments, the transition from the first amplification to the second amplification is performed 11 days after the disruption.

In some embodiments, the TIL is not stored after the first amplification and before the rapid second amplification, and the TIL is directly subjected to the second amplification (e.g., in some embodiments, not stored during the transition from step B to step D as shown in fig. 1). In some embodiments, the transition is performed in a closed system as described herein. In some embodiments, TIL from the first amplification (second population of TILs) is directly subjected to the second amplification without a transition phase.

In some embodiments, the transition from the first amplification to the second amplification (e.g., according to step C of fig. 1) is performed in a closed system bioreactor. In some embodiments, the TIL amplification as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor.

D. Step D: second amplification

In some embodiments, the number of TIL cell populations is expanded after collection and initial batch processing, e.g., after step a and step B and the transition referred to as step C, as indicated in fig. 1. This further amplification is referred to herein as a second amplification, which may include an amplification process commonly referred to in the art as a rapid amplification process (REP); and a process as indicated in step D of fig. 1. The second amplification is typically accomplished in a gas-permeable vessel using a medium comprising a plurality of components, including feeder cells, a cytokine source, and anti-CD 3 antibodies.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process as indicated in step D of FIG. 1) may be performed using any TIL culture flask or vessel known to those of skill in the art. In some embodiments, the second TIL amplification may be performed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL amplification may be performed for about 7 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 8 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 9 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 10 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 11 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 12 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 13 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 14 days.

In some embodiments, the second amplification may be performed in a gas-permeable container using the methods of the present disclosure (including, for example, amplification known as REP; and the process as indicated in step D of FIG. 1). For example, TIL can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators may include, for example, anti-CD 3 antibodies, such as about 30ng/mL OKT3, mouse monoclonal anti-CD 3 antibodies (available from Ortho-McNeil, laritan, N.J., or Meitian Biotechnology, inc. of Ornith, calif.), or UHCT-1 (available from BioLegend, san Diego, calif., U.S.A.). TIL can be amplified by including more than one antigen of cancer (including antigenic portions thereof, e.g., epitopes) during the second amplification to induce further in vitro stimulation of TIL, which antigen can optionally be expressed by a vector, e.g., human leukocyte antigen A2 (HLa-A2) binding peptide, e.g., 0.3 μ MMART-1:26-35 (27L) or gpl 00:209-217 (210M), optionally in the presence of T cell growth factors (e.g., 300IU/mL IL-2 or IL-15). Other suitable antigens may include, for example, NY-ESO-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TIL can also be rapidly amplified by restimulation with the same cancer antigen pulsed onto antigen presenting cells expressing HLA-A 2. Alternatively, the TIL may be further restimulated with, for example, example irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation is performed as part of the second amplification. In some embodiments, the second expansion is performed in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000IU/mL, about 1500IU/mL, about 2000IU/mL, about 2500IU/mL, about 3000IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, or about 8000IU/mL IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000IU/mL, between 2000 and 3000IU/mL, between 3000 and 4000IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000IU/mL, between 6000 and 7000IU/mL, between 7000 and 8000IU/mL, or 8000IU/mL of IL-2.

In some embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1. Mu.g/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises OKT-3 antibodies between 0.1ng/mL and 1ng/mL, between 1ng/mL and 5ng/mL, between 5ng/mL and 10ng/mL, between 10ng/mL and 20ng/mL, between 20ng/mL and 30ng/mL, between 30ng/mL and 40ng/mL, between 40ng/mL and 50ng/mL, and between 50ng/mL and 100 ng/mL. In some embodiments, the cell culture medium does not comprise an OKT-3 antibody. In some embodiments, the OKT-3 antibody is Moromolizumab.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is employed as the combination during the second amplification. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included during the second amplification, including, for example, during the step D process according to FIG. 1 and described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed as the combination during the second amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the step D process according to FIG. 1 and as described herein.

In some embodiments, the second expansion may be performed in a supplemented cell culture medium comprising IL-2, OKT-3, antigen presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion is performed in supplemented cell culture medium. In some embodiments, the supplemental cell culture medium comprises IL-2, OKT-3 and antigen presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3 and antigen presenting cells (APC; also referred to as antigen presenting feeder cells). In some embodiments, the second expansion is performed in a cell culture medium comprising IL-2, OKT-3 and antigen presenting feeder cells (i.e., antigen presenting cells).

In some embodiments, the second amplification medium comprises about 500IU/mL IL-15, about 400IU/mL IL-15, about 300IU/mL IL-15, about 200IU/mL IL-15, about 180IU/mL IL-15, about 160IU/mL IL-15, about 140IU/mL IL-15, about 120IU/mL IL-15, or about 100IU/mL IL-15. In some embodiments, the second amplification medium comprises about 500IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the second amplification medium comprises about 400IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the second amplification medium comprises about 300IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the second amplification medium comprises about 200IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the second amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mLIL-21. In some embodiments, the second amplification medium comprises about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the second amplification medium comprises about 2IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In some embodiments, in the rapid expansion and/or the second expansion, the ratio of TIL to PBMCs and/or antigen presenting cells is about 1: 25. about 1: 50. about 1: 100. about 1: 125. about 1: 150. about 1: 175. about 1: 200. about 1: 225. about 1: 250. about 1: 275. about 1: 300. about 1: 325. about 1: 350. about 1: 375. about 1:400 or about 1:500. in some embodiments, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1:50 and 1: 300. In some embodiments, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1:100 and 1: 200.

In some embodiments, REP and/or second expansion is performed in a culture flask, wherein bulk TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30mg/mL OKT3 anti-CD 3 antibody, and 3000IU/mL IL-2 in 150mL medium. Media exchange (2/3 media exchange with fresh media, typically by aspiration) is performed until cells are transferred to the alternate growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable containers, as discussed more fully below.

In some embodiments, the second amplification (which may include a process called the REP process) is shortened to 7 to 14 days, as described in the examples and illustrations. In some embodiments, the second amplification is shortened to 11 days.

In some embodiments, REP and/or second amplification may be performed using a T-175 flask and a gas permeable bag (Tran et al, J. Immunotherapy.) "2008,31,742-51; dudley et al, J. Immunotherapy. 2003,26,332-42) or a gas permeable culture vessel (G-REX flask) as previously described. In some embodiments, the second amplification (including amplification called rapid amplification) is performed in T-175 flasks, and about 1X 10 ⁶ TILs suspended in 150mL of medium may be added to each T-175 flask. TIL can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000IU/mL IL-2 and 30ng/mL anti-CD 3. T-175 flasks can be incubated at 37℃with 5% CO ₂. Half of the medium can be replaced on day 5 with 50/50 medium with 3000IU/mL IL-2. In some embodiments, cells from two T-175 flasks may be pooled in a 3L bag on day 7, and 300mL AIM V with 5% human AB serum and 3000IU/mL IL-2 added to 300mL TIL suspension. The number of cells in each bag was counted daily or every two days, fresh medium was added to keep the cell count between 0.5 and 2.0X10 ⁶ cells/mL.

In some embodiments, the second amplification (which may include amplification called REP, as well as those mentioned in step D of FIG. 1) may be performed in 500mL capacity gas-permeable flasks (G-REX 100, available from Wilson wolf manufacturing company (Wilson Wolf Manufacturing Corporation) of New Briton, minnesota) with a 100cm gas-permeable bottom, 5X 10 ⁶ or 10X 10 ⁶ TILs may be cultured with PBMC in 400mL 50/50 medium supplemented with 5% human AB serum, 3000IU/mL IL-2, and 30ng/mL anti-CD 3 (OKT 3). G-REX-100 flasks can be incubated at 37℃in 5% CO ₂. On day 5, 250mL of supernatant may be removed, placed in a centrifuge bottle, and centrifuged at 1500rpm (491 Xg) for 10 minutes. The TIL pellet can be resuspended in 150mL fresh medium with 5% human AB serum, 3000IU/mL IL-2 and added back to the original G-REX 100 flask. When TIL was continuously expanded in G-REX 100 flasks, at day 7, TIL in each G-REX 100 was suspended in 300mL of medium present in each flask and the cell suspension was split into 3 100mL aliquots that were used to inoculate 3G-REX 100 flasks. 150mL of AIM-V with 5% human AB serum and 3000IU/mL IL-2 can then be added to each flask. G-REX 100 flasks can be incubated at 37℃with 5% CO ₂ and after 4 days 150mL AIM-V with 3000IU/mL IL-2 can be added to each G-REX 100 flask. Cells may be harvested on day 14 of culture.

In some embodiments, the second amplification (including amplification called REP) is performed in culture flasks, wherein the bulk TIL is mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30mg/mL OKT3 anti-CD 3 antibody, and 3000IU/mL IL-2 in 150mL of medium. In some embodiments, the medium is replaced until the cells are transferred to the replacement growth chamber. In some embodiments, 2/3 of the medium is replaced with fresh medium by aspiration. In some embodiments, the replacement growth chamber includes a G-REX flask and a gas-permeable container, as discussed more fully below.

In some embodiments, a second amplification (including an amplification called REP) is performed, which further includes a step in which TIL with excellent tumor reactivity is selected. Any selection method known in the art may be used. For example, the method described in U.S. patent application publication 2016/0010058A1, the disclosure of which is incorporated herein by reference, may be used to select TILs with excellent tumor reactivity.

Alternatively, a cell viability (availability) assay may be performed after a second amplification, including an amplification known as REP amplification, using standard assays known in the art. For example, trypan blue exclusion analysis can be performed on bulk TIL samples, which selectively marks dead cells and allows survival assessment. In some embodiments, the TIL samples may be calculated and survival determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience of lorens, ma). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, the second amplification of TIL (including amplification called REP) may be performed using a T-175 flask and a gas permeable bag (Tran et al, 2008, J.Immunotherapy, 31,742-751, and Dudley et al, 2003, J.Immunotherapy, 26, 332-342) or a gas permeable G-REX flask as previously described. In some embodiments, the second amplification is performed using a culture flask. In some embodiments, the second amplification is performed using a gas-permeable G-REX flask. In some embodiments, the second amplification is performed in T-175 flasks, and about 1X 10 ⁶ TILs are suspended in about 150mL of medium and added to each T-175 flask. TIL was incubated with irradiated (50 Gy) allogeneic PBMC as "feeder" cells at a ratio of 1:100, and cells were cultured in a 1:1 mixture (50/50 medium) supplemented with 3000IU/mL IL-2 and 30ng/mL anti-CD 3 CM and AIM-V medium. T-175 flasks were incubated at 37℃with 5% CO ₂. In some embodiments, half of the medium is replaced on day 5 with 50/50 medium with 3000IU/mL IL-2. In some embodiments, on day 7, cells from 2T-175 flasks were pooled in a 3L bag and 300mL AIM-V with 5% human AB serum and 3000IU/mL IL-2 was added to 300mL TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh medium can be added to maintain the cell count between about 0.5 and about 2.0X10 ⁶ cells/mL.

In some embodiments, the second amplification (including that referred to as REP) is performed in 500mL of culture flasks (G-REX-100, wilson Wolf) with a 100cm ² permeable bottom, and about 5X 10 ⁶ or 10X 10 ⁶ TILs are cultured with irradiated allogeneic PBMC at a 1:100 ratio in 400mL of 50/50 medium supplemented with 3000IU/mL IL-2 and 30ng/mL anti-CD 3. G-REX-100 flasks were incubated at 37℃with 5% CO ₂. In some embodiments, on day 5, 250mL of supernatant is removed and placed in a centrifuge bottle and centrifuged at 1500rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150mL fresh 50/50 medium with 3000IU/mL IL-2 and added back to the original G-REX-100 flask. In the example where TIL was continuously amplified in G-REX-100 flasks, on day 7, TIL in each G-REX-100 was suspended in 300mL of medium present in each flask and the cell suspension was split into three 100mL aliquots that were used to inoculate 3G-REX-100 flasks. 150mL of AIM-V with 5% human AB serum and 3000IU/mL IL-2 was then added to each flask. G-REX-100 flasks were incubated at 37℃with 5% CO ₂ and after 4 days 150mL AIM-V with 3000IU/mL IL-2 was added to each G-REX-100 flask. Cells were collected on day 14 of culture.

The various antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene segments: v (variable region), D (variable region), J (junction region) and C (constant region) determine the binding specificity and downstream application of immunoglobulins to T Cell Receptors (TCRs). The present invention provides a method for producing TIL that exhibits and increases T cell reservoir diversity. In some embodiments, the TIL obtained by the methods of the invention exhibits increased T cell reservoir diversity. In some embodiments, the TIL obtained in the second expansion exhibits increased T cell reservoir diversity. In some embodiments, increasing diversity is increasing immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in the immunoglobulin and in the heavy chain of the immunoglobulin. In some embodiments, the diversity is present in the immunoglobulin and in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T Cell Receptor (TCR) α and/or β is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, TCRab (i.e., tcra/β) is expressed in increased.

In some embodiments, the second expansion medium (e.g., sometimes referred to as CM2 or a second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs) as discussed in more detail below.

In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS ^TMOpTmizer^TM T cell expansion basal medium, CTS ^TMOpTmizer^TM T cell expansion SFM, CTS ^TM AIM-V medium, CTS ^TMAIM-V SFM、LymphoONE^TM T cell expansion no Xeno medium, darby's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), isagl Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (αMEM), grasgang minimal essential medium (G-MEM), RPMI growth medium, and Iskov modified Darby's medium.

In some embodiments, the serum supplement or serum replacement includes (but is not limited to) one or more of the following: CTS ^TM OpTmizer T cell-expanded serum supplement, CTS ^TM immune cell serum replacement, one or more albumin or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and compounds containing trace element moieties Ag⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺ and Zr ⁴⁺. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, the CTS ^TMOpTmizer^TM T cell immune cell serum replacement is used with conventional growth media including, but not limited to, CTS ^TMOpTmizer^TM T cell expansion basal media, CTS ^TMOpTmizer^TM T cell expansion SFM, CTS ^TM AIM-V media, CST ^TMAIM-V SFM、LymphoONE^TM T cell expansion no Xeno media, darburg Modified Eagle's Medium (DMEM), minimal Essential Media (MEM), eagle Basal Media (BME), RPMI 1640, F-10, F-12, minimal essential media (αmem), glasgang minimal essential media (G-MEM), RPMI growth media, and iskioskov modified darburg.

In some embodiments, the defined medium is CTS ^TMOpTmizer^TM T cell expansion SFM (sammer feishier technology). Any CTS ^TMOpTmizer^TM formulation may be used in the present invention. CTS ^TMOpTmizer^TM T cell expansion SFM is a combination of 1L CTS ^TMOpTmizer^TM T cell expansion basal medium and 26mL CTS ^TMOpTmizer^TM T cell expansion supplement mixed together prior to use. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), the final concentration of 2-mercaptoethanol in the medium is 55 μm.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine at a concentration of about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5mM (i.e.,). In some embodiments, the serum-free medium or defined medium is supplemented with glutamine at a concentration of about 2mM (i.e./>)。

In some embodiments, the non-trace element fraction ingredients in the defined ingredient medium are present in the concentration ranges listed in the column entitled "concentration ranges in 1X medium" in table 12. In other embodiments, the non-trace element fraction ingredients in the defined ingredient medium are present at the final concentrations listed in the column entitled "preferred embodiment of Medium" 1X Medium in Table 12. In other embodiments, the defined medium is a basal cell medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace amounts of ingredients of the types and concentrations listed in table 12 and column entitled "preferred embodiments of supplement".

In some embodiments, the defined medium described in Smith et al, clinical and transformation immunology 4 (1) 2015 (doi: 10.1038/cti.2014.31) is suitable for use in the present invention. Briefly, RPMI or CTS ^TMOpTmizer^TM was used as basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS ^TM immune cell serum replacement.

In some embodiments, the second amplification is performed in a closed system bioreactor, for example according to step D of fig. 1. In some embodiments, the TIL amplification as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the step of rapid amplification or second amplification is split into multiple steps to achieve a vertical expansion of the culture scale (scale up) by: (a) Rapid amplification or second amplification by culturing TIL in a small scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 3 days to 7 days; and (b) effecting transfer of the TIL in the small-scale culture to a second vessel (e.g., G-REX-500-MCS vessel) larger than the first vessel, culturing the TIL from the small-scale culture in the larger-scale culture in the second vessel for a period of about 4 days to 7 days.

In some embodiments, the step of rapid amplification or second amplification is split into multiple steps to achieve lateral expansion of the culture scale (scale out) by: (a) Performing rapid amplification or second amplification by culturing TIL in a first small-scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 3 days to 7 days; and (b) effecting transfer and distribution of the TIL from the first small scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second vessels of equal size to the first vessel, in each of which second vessels the fraction of TIL from the first small scale culture transferred into such second vessel is cultured in the second small scale culture for a period of about 4 days to 7 days.

In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2 to 5 TIL subpopulations.

In some embodiments, the step of rapid amplification or second amplification is split into multiple steps to achieve culture scale-up laterally and scale-up longitudinally by: (a) Performing rapid-scale or second amplification by culturing TIL in a small-scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 3 days to 7 days; and (b) effecting transfer and distribution of TIL from the small scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second vessels (e.g., G-REX-500MCS vessels) of larger size than the first vessel, in each of which second vessels the TIL fraction from the small scale culture transferred to such second vessel is cultured in the larger scale culture for a period of about 4 days to 7 days.

In some embodiments, the step of rapid amplification or second amplification is split into multiple steps to achieve culture scale-up laterally and scale-up longitudinally by: (a) Rapid amplification or second amplification by culturing TIL in a small scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 5 days; and (b) effecting transfer and distribution of the TIL from the small-scale culture into 2,3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which the TIL fraction from the small-scale culture transferred to such second container is cultured in the larger-scale culture for a period of about 6 days.

In some embodiments, each second container comprises at least 10 ⁸ TILs during rapid amplification or second amplification split. In some embodiments, each second container comprises at least 10 ⁸ TILs, at least 10 ⁹ TILs, or at least 10 ¹⁰ TILs upon rapid amplification or second amplification split. In one exemplary embodiment, each second container contains at least 10 ¹⁰ TILs.

In some embodiments, the first small-scale TIL culture is partitioned into multiple subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2 to 5 subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2, 3, 4, or 5 subpopulations.

In some embodiments, after rapid amplification or second amplification is completed, the multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid amplification or second amplification is completed, more than one subpopulation of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, each TIL subpopulation comprises a therapeutically effective amount of TIL after rapid amplification is completed.

In some embodiments, the rapid amplification or the second amplification is performed for a period of about 3 to 7 days prior to splitting into multiple steps. In some embodiments, the diversion of the rapid amplification or the second amplification is performed about day 3, day 4, day 5, day 6, or day 7 after the initiation of the rapid amplification or the second amplification.

In some embodiments, the rapid amplification or the split of the second amplification is performed about day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15 or day 16, day 17 or day 18 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary embodiment, the rapid amplification or the split of the second amplification is performed about day 16 after the start of the first amplification.

In some embodiments, after the splitting, the rapid amplification or the second amplification is further performed for a period of about 7 to 11 days. In some embodiments, after the split, the rapid amplification or the second amplification is further performed for a period of about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the cell culture medium used for rapid expansion or second expansion prior to the split comprises the same components as the cell culture medium used for rapid expansion or second expansion after the split. In some embodiments, the cell culture medium used for rapid expansion or second expansion prior to the splitting comprises a different composition than the cell culture medium used for rapid expansion or second expansion after the splitting.

In some embodiments, the cell culture medium for rapid expansion or second expansion prior to diversion comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion or second expansion prior to diversion comprises IL-2, OKT-3 and further optional APC. In some embodiments, the cell culture medium for rapid expansion or second expansion prior to diversion comprises IL-2, OKT-3 and APC.

In some embodiments, the cell culture medium for rapid expansion or second expansion prior to splitting is produced by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion or second expansion prior to splitting is produced by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, OKT-3 and APC. In some embodiments, the cell culture medium used for rapid expansion or second expansion prior to splitting is produced by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid expansion or second expansion prior to splitting is produced by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3 and APC.

In some embodiments, the cell culture medium used for rapid expansion or second expansion after fractionation comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion or second expansion after fractionation comprises IL-2 and OKT-3. In some embodiments, the cell culture medium for rapid expansion or second expansion after fractionation is produced by replacing the cell culture medium for rapid expansion or second expansion before fractionation with fresh medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium for rapid expansion or second expansion after fractionation is produced by replacing the cell culture medium for rapid expansion or second expansion before fractionation with fresh medium comprising IL-2 and OKT-3.

In some embodiments, the rapid amplification split is performed in a closed system.

In some embodiments, the longitudinal expansion of the TIL culture scale during rapid expansion or second expansion includes adding fresh cell culture medium (also referred to as feeding TIL) to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture frequently. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL by a constant flow. In some embodiments, rapid expansion and feeding is performed using an automated cell expansion system such as Xuri W.

1. Feeder cells and antigen presenting cells

In some embodiments, the second amplification procedure described herein (e.g., including, for example, the amplification described in step D of fig. 1, as well as the amplification referred to as REP) requires an excess of feeder cells during the REP TIL amplification and/or during the second amplification. In many embodiments, the feeder cells are obtained from Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units of healthy blood donors. PBMC are obtained using standard methods, such as Ficoll-Paque gradient separation.

In general, allogeneic PBMCs are inactivated by irradiation or heat treatment, as described in the examples for the REP procedure, which provides an exemplary protocol for assessing replication incompetence of irradiated allogeneic PBMCs.

In some embodiments, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedure described herein if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of second expansion (i.e., the starting day of second expansion).

In some embodiments, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedure described herein if the total number of living cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 is not increased compared to the initial number of living cells placed in culture on day 0 of REP and/or day 0 of second expansion (i.e., the starting day of second expansion). In some embodiments, PBMC are cultured in the presence of 30ng/mL OKT3 antibody and 3000IU/mL IL-2.

In some embodiments, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedure described herein if the total number of living cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 is not increased compared to the initial number of living cells placed in culture on day 0 of REP and/or day 0 of second expansion (i.e., the starting day of second expansion). In some embodiments, PBMC are cultured in the presence of 5 to 60ng/mL OKT3 antibody and 1000 to 6000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 10 to 50ng/mL OKT3 antibody and 2000 to 5000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 20 to 40ng/mL OKT3 antibody and 2000 to 4000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 25 to 35ng/mL OKT3 antibody and 2500 to 3500IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is about 1: 25. about 1: 50. about 1: 100. about 1: 125. about 1: 150. about 1: 175. about 1: 200. about 1: 225. about 1: 250. about 1: 275. about 1: 300. about 1: 325. about 1: 350. about 1: 375. about 1:400 or about 1:500. in some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1: 300. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1: 200.

In some embodiments, the second expansion procedure described herein requires a ratio of about 2.5X10 ⁹ feeder cells to about 100X 10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of about 2.5X10 ⁹ feeder cells to about 50X 10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of about 2.5X10 ⁹ feeder cells to about 25X 10 ⁶ TILs.

In some embodiments, the second amplification procedure described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are obtained from Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units of healthy blood donors. PBMC are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aapcs) are used in place of PBMCs.

Generally, allogeneic PBMCs are inactivated by irradiation or heat treatment for the TIL amplification procedures described herein, including the exemplary procedures described in the figures and examples.

In some embodiments, artificial antigen presenting cells are used in the second expansion in place of or in combination with PBMCs.

2. Cytokines and other additives

Or combinations of cytokines for rapid amplification and/or second amplification of TIL are also possible, as described in U.S. patent application publication No. US2017/0107490 A1, using two or more of the IL-2, IL-15 and IL-21 combinations, the disclosures of which are incorporated herein by reference. Thus, it is possible to combine IL-2 with IL-15, IL-2 with IL-21, IL-15 with IL-21, and IL-2, IL-15 with IL-21, the latter having particular utility in many embodiments. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, in particular T cells as described therein.

In some embodiments, step D may also comprise adding OKT-3 antibody or moromiab to the medium, as described elsewhere herein. In some embodiments, step D may also include adding a 4-1BB agonist to the medium, as described elsewhere herein. In some embodiments, step D can also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. Furthermore, additives such as peroxisome proliferator activated receptor gamma coactivator I-alpha agonists, including proliferator activated receptor (PPAR) -gamma agonists, such as thiazolidinedione compounds, may be used in the medium during step D, as described in US patent application publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

E. step E: collecting TIL

After the second expansion step, the cells may be collected. In some embodiments, TIL is collected after one, two, three, four or more amplification steps, such as provided in fig. 1. In some embodiments, TIL is collected after two amplification steps, such as provided in fig. 1.

The TIL may be collected in any suitable and sterile manner, including, for example, centrifugation. Methods for collecting TIL are well known in the art and any such known methods may be used with the process of the present invention. In some embodiments, the TIL is collected using an automated system.

Cell collectors and/or cell processing systems are available from a variety of sources, including, for example, fei Senyou s Kabi (Fresenius Kabi), tomtec LIFE SCIENCE, perkin elmer (PERKIN ELMER) and Inotech Biosystems International, inc. Any cell-based collector may be used in the methods of the invention. In some embodiments, the cell collector and/or the cell processing system is a membrane-based cell collector. In some embodiments, cell collection is performed by a cell processing system, such as the LOVO system (manufactured by Fei Senyou SICAR). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any vendor that can pump a solution containing cells through a membrane or filter (e.g., a rotating membrane or rotating filter) in a sterile and/or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell collector and/or cell processing system may perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed sterile system.

In some embodiments, the collecting, e.g., according to step E of fig. 1, is performed in a closed system bioreactor. In some embodiments, the TIL amplification as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, step E according to fig. 1 is performed according to the process described herein. In some embodiments, the containment system is accessed through a syringe under sterile conditions to maintain sterility and containment properties of the system. In some embodiments, a closed system as described in the examples is employed.

In some embodiments, the TIL is collected according to the methods described in the examples. In some embodiments, the TIL between day 1 and day 11 is collected using the methods described in the steps mentioned herein, e.g., day 11 TIL collection in the examples. In some embodiments, the TIL between day 12 and 24 is collected using the methods described in the steps mentioned herein, e.g., day 22 TIL collection in the examples. In some embodiments, the TIL between day 12 and day 22 is collected using the methods described in the steps mentioned herein, e.g., day 22 TIL collection in the examples.

F. Step F: final formulation and transfer to infusion container

After steps a through E, provided in an exemplary order as in fig. 1 and as detailed above and herein, are completed, the cells are transferred into a container for administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a sufficient number of TILs are obtained for treatment using the amplification methods described above, they are transferred to a container for administration to a patient.

In some embodiments, TIL amplified using APCs of the present disclosure is administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in sterile buffer. TIL amplified using PBMCs of the present disclosure may be administered by any suitable route known in the art. In some embodiments, the T cells are administered in the form of a single intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Gen 3TIL manufacturing Process

Without being bound by any particular theory, it is believed that the initial first expansion that initiates T cell activation and the subsequent rapid second expansion that enhances T cell activation as described in the methods of the invention allow for the preparation of expanded T cells that retain a "younger" phenotype, thus it is expected that the expanded T cells of the invention may exhibit higher cytotoxicity to cancer cells than T cells expanded by other methods. In particular, it is believed that activation of T cells by exposure to anti-CD 3 antibodies (e.g., OKT-3), IL-2, and optionally Antigen Presenting Cells (APCs) as taught by the methods of the invention is initiated and then enhanced by subsequent exposure to additional anti-CD 3 antibodies (e.g., OKT-3), IL-2, and APCs, which limit or avoid maturation of T cells in the medium, thereby producing a population of T cells with a less mature phenotype that is less depleted by culture expansion and exhibits higher cytotoxicity to cancer cells. In some embodiments, the step of rapid second amplification is split into multiple steps to achieve a longitudinal expansion of the culture scale by: (a) Rapid second expansion by culturing T cells in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 days to 4 days; and (b) effecting transfer of the T cells in the small-scale culture to a second vessel (e.g., a G-REX 500MCS vessel) that is larger than the first vessel, culturing T cells from the small-scale culture in the larger-scale culture in the second vessel for a period of about 4 days to 7 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale by: (a) Performing a rapid second expansion by culturing T cells in a first small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 days to 4 days; and then (b) transferring and dispensing T cells from the first small scale culture into at least 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers of the same size as the first container, wherein in each second container, T cell fractions from the first small scale culture transferred to such second container are cultured in the second small scale culture for a period of about 4 days to 7 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Rapid second expansion by culturing T cells in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 days to 4 days; and then (b) transferring and dispensing T cells from the small scale culture into at least 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX 500MCS containers) of larger size than the first container, in each of which the T cell fraction from the first small scale culture transferred to such second container is cultured in the larger scale culture for a period of about 4 days to 7 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Rapid second expansion by culturing T cells in a small scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 4 days; and (b) transferring and dispensing T cells from the small scale culture into 2,3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which the T cell fraction from the first small scale culture transferred to such second container is cultured in the larger scale culture for a period of about 5 days.

In some embodiments, each second container comprises at least 10 ⁸ TILs at the time of rapid amplification split. In some embodiments, each second container comprises at least 10 ⁸ TILs, at least 10 ⁹ TILs, or at least 10 ¹⁰ TILs at the time of rapid amplification split. In one exemplary embodiment, each second container contains at least 10 ¹⁰ TILs.

In some embodiments, after rapid amplification is completed, the multiple subpopulations contain a therapeutically effective amount of TIL. In some embodiments, after rapid amplification is completed, more than one subpopulation of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, each TIL subpopulation comprises a therapeutically effective amount of TIL after rapid amplification is completed.

In some embodiments, rapid amplification is performed for a period of about 1 to 5 days prior to separation into multiple steps. In some embodiments, the diversion of rapid amplification is performed about day 1, day 2, day 3, day 4, or day 5 after the start of rapid amplification.

In some embodiments, the diversion of rapid amplification is performed about day 8, day 9, day 10, day 11, day 12, or day 13 after the start of the first amplification (i.e., pre-REP amplification). In one exemplary embodiment, the rapid amplification split is performed about day 10 after initiation of the first amplification. In another exemplary embodiment, the rapid amplification split is performed about day 11 after the initiation of the first amplification.

In some embodiments, rapid amplification is further performed for a period of about 4 to 11 days after the diversion. In some embodiments, after the split, the rapid amplification is further performed for a period of about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the cell culture medium for rapid expansion prior to fractionation comprises the same components as the cell culture medium for rapid expansion after fractionation. In some embodiments, the cell culture medium for rapid expansion prior to fractionation comprises a different composition than the cell culture medium for rapid expansion after fractionation.

In some embodiments, the cell culture medium for rapid expansion prior to fractionation comprises IL-2, optionally OKT-3 and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to diversion comprises IL-2, OKT-3 and further optional APC. In some embodiments, the cell culture medium for rapid expansion prior to fractionation comprises IL-2, OKT-3 and APC.

In some embodiments, the cell culture medium for rapid expansion prior to splitting is produced by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is produced by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, OKT-3 and APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is produced by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid expansion prior to splitting is produced by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3 and APC.

In some embodiments, the cell culture medium for rapid expansion after fractionation comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium for rapid expansion after fractionation comprises IL-2 and OKT-3. In some embodiments, the cell culture medium for rapid expansion after fractionation is produced by replacing the cell culture medium for rapid expansion before fractionation with fresh medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium for rapid expansion after fractionation is produced by replacing the cell culture medium for rapid expansion before fractionation with fresh medium comprising IL-2 and OKT-3.

In some embodiments, the rapidly amplified split stream is performed in a closed system.

In some embodiments, the longitudinal expansion of the TIL culture scale during rapid expansion includes adding fresh cell culture medium (also referred to as feeding TIL) to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture frequently. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL by a constant flow. In some embodiments, rapid expansion and feeding is performed using an automated cell expansion system such as Xuri W.

In some embodiments, the rapid second expansion occurs after T cell activation achieved by initiating the first expansion begins to decrease, slow, decline or regress.

In some embodiments, the rapid second expansion is performed after T cell activation by initiating the first expansion has been reduced by just or about (at or about)1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99 or 100%.

In some embodiments, the rapid second expansion occurs after T cell activation achieved by initiating the first expansion has been reduced by just or a percentage in the range of about 1% to 100%.

In some embodiments, the rapid second expansion occurs after T cell activation achieved by initiating the first expansion has been reduced by just or about a percentage in the range of 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

In some embodiments, the rapid second expansion is performed after T cell activation by initiating the first expansion has been reduced by at least just or about 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98 or 99%.

In some embodiments, the rapid second expansion is performed after T cell activation achieved by initiating the first expansion has been reduced by at most just or about 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99 or 100%.

In some embodiments, the decrease in T cell activation achieved by initiating the first expansion is determined by a decrease in the amount of interferon gamma released by the T cells in response to antigen stimulation.

In some embodiments, the initial first expansion of T cells is performed for a period of up to just or about 7 days or about 8 days.

In some embodiments, the initial first expansion of T cells is performed for a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

In some embodiments, the initial first expansion of T cells is performed for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

In some embodiments, the rapid second expansion of T cells is performed for a period of up to just or about 11 days.

In some embodiments, the rapid second expansion of T cells is performed for a period of up to just or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or 11 days.

In some embodiments, the rapid second expansion of T cells is performed for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the initial first expansion of T cells is performed for a period of time of from just or about 1 day to just or about 7 days and the rapid second expansion of T cells is performed for a period of time of from just or about 1 day to just or about 11 days.

In some embodiments, the initial first expansion of T cells is performed for a period of up to or about 1,2, 3, 4, 5, 6, 7, or 8 days, and the rapid second expansion of T cells is performed for a period of up to or about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

In some embodiments, the initial first expansion of T cells is performed for a period of time of from just or about 1 day to just or about 8 days, and the rapid second expansion of T cells is performed for a period of time of from just or about 1 day to just or about 9 days.

In some embodiments, the initial first expansion of T cells is performed for a period of 8 days, and the rapid second expansion of T cells is performed for a period of 9 days.

In some embodiments, the initial first expansion of T cells is performed for a period of time of from just or about 1 day to at or about 7 days, and the rapid second expansion of T cells is performed for a period of time of from at or about 1 day to at or about 9 days.

In some embodiments, the initial first expansion of T cells is performed for a period of 7 days and the rapid second expansion of T cells is performed for a period of 9 days.

In some embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL).

In some embodiments, the T cells are bone Marrow Infiltrating Lymphocytes (MILs).

In some embodiments, the T cells are Peripheral Blood Lymphocytes (PBLs).

In some embodiments, the T cells are obtained from a donor having cancer.

In some embodiments, the T cells are TILs obtained from tumors resected from patients with cancer.

In some embodiments, the T cell is MILs obtained from bone marrow of a patient having a hematopoietic malignancy.

In some embodiments, the T cells are peripheral blood mononuclear cells (PBLs) obtained from a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is a cancer selected from the group consisting of: melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematopoietic malignancy.

In certain aspects of the invention, immune effector cells (e.g., T cells) can be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL isolation). In a preferred aspect, cells from the circulating blood of the subject are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulosa cells, B cells, other nucleated leukocytes, erythrocytes and platelets. In one aspect, cells collected by single harvest may be washed to remove the plasma fraction, optionally placing the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In an alternative embodiment, the wash solution lacks calcium, possibly magnesium, or possibly many, if not all, divalent cations. In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the erythrocytes and depleting monocytes, e.g., by PERCOLL gradient centrifugation or by countercurrent centrifugation elution.

In some embodiments, the T cells are PBLs isolated from whole donor blood or lymphocyte-enriched apheresis products. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the following: melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematopoietic malignancy. In some embodiments, the PBLs are isolated from whole blood or lymphocyte-enriched apheresis product using positive or negative selection methods, i.e., using a marker of T cell phenotype (e.g., cd3+cd45+) to remove the PBLs, or removing non-T cell phenotype cells to leave the PBLs. In other embodiments, the PBLs are separated by gradient centrifugation. After isolation of the PBLs from the donor tissue, initial first amplification of the PBLs may begin according to the initial first amplification step of any of the methods described herein by inoculating an appropriate number of isolated PBLs (in some embodiments, about 1 x 10 ⁷ PBLs) into the initial first amplification culture.

An exemplary TIL process, referred to herein as process 3 (also referred to as Gen 3), containing some of these features is depicted in fig. 8 (particularly, e.g., fig. 8B and/or 8C and/or 8D), and some of the advantages of this embodiment of the invention in Gen 2 are depicted in fig. 1, 2, 8, 30 and 31 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). Embodiments of Gen 3 are shown in fig. 1, 8, and 30 (specifically, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). Process 2A or Gen 2A is also described in U.S. patent publication No. 2018/0280436, which is incorporated herein by reference in its entirety. The Gen 3 process is also described in International patent publication WO 2020/096988.

As described herein and generally summarized, TILs are taken from patient samples, and the TIL amplification procedure described herein and referred to as Gen 3 is used to amplify the number thereof prior to transplantation into a patient. In some embodiments, the TIL may optionally be genetically manipulated as described below. In some embodiments, the TIL may be cryopreserved before or after amplification. After thawing, it may also be re-stimulated to increase its metabolism prior to infusion into a patient.

In some embodiments, the initial first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 1 to 8 days, and the rapid second amplification (including the process referred to herein as rapid amplification protocol (REP), and the process shown as step D in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 1 to 9 days, as discussed in detail below and in the examples and illustrations. In some embodiments, the initial first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 1 to 8 days, and the rapid second amplification (including the process referred to herein as rapid amplification protocol (REP), and the process shown as step D in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 1 to 8 days, as discussed in detail below and in the examples and illustrations. In some embodiments, the initial first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 1 to 7 days, and the rapid second amplification (including the process referred to herein as rapid amplification protocol (REP), and the process shown as step D in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 1 to 9 days, as discussed in detail below and in the examples and illustrations. In some embodiments, the initial first amplification (including the process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 8 (particularly, e.g., fig. 1B and/or fig. 8C)) is 1 to 7 days, and the rapid second amplification (including the process referred to herein as rapid amplification protocol (REP), and the process shown as step D in fig. 8 (particularly, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D)) is 1 to 10 days, as discussed in detail below and in the examples and illustrations. In some embodiments, the initial first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 8 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 7 to 9 days. In some embodiments, the first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 8 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 8 to 9 days. In some embodiments, the initial first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 7 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 7 to 8 days. In some embodiments, the first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 8 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 8 days. In some embodiments, the first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 8 days and the rapid second amplification (e.g., the amplification described as step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 9 days. In some embodiments, the first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 8 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is initiated for 10 days. In some embodiments, the initial first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 7 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 7 to 10 days. In some embodiments, the initial first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 7 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 8 to 10 days. In some embodiments, the initial first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 7 days and the rapid second amplification (e.g., the amplification described as step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 9 to 10 days. In some embodiments, the initial first amplification (e.g., the amplification described as step B in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is shortened to 7 days and the rapid second amplification (e.g., the amplification as described in step D in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is 7 to 9 days. In some embodiments, the combination of the initial first amplification and the rapid second amplification (e.g., described as step B and step D amplification in fig. 8 (e.g., particularly fig. 1B and/or fig. 8C)) is 14 to 16 days, as discussed in detail below and in the examples and illustrations. In particular, certain embodiments of the invention are believed to comprise an initial first amplification step in which the TIL is activated by exposure to an anti-CD 3 antibody (e.g., OKT-3) in the presence of IL-2 or to an antigen in the presence of at least IL-2 and an anti-CD 3 antibody (e.g., OKT-3). In certain embodiments, the TIL activated in the initial first amplification step as described above is a first population of TILs, i.e., it is a population of primary cells.

The "step" designation A, B, C, etc. hereinafter refers to a non-limiting example in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) and to certain non-limiting embodiments described herein. The following and sequences of steps in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) are exemplary, and the application and methods disclosed herein contemplate any combination or sequence of steps, as well as additional steps, step repetitions, and/or step omissions.

A. Step A: obtaining a patient tumor sample

Typically, TILs are initially obtained from a patient tumor sample ("primary TIL") or from circulating lymphocytes (e.g., peripheral blood lymphocytes, including peripheral blood lymphocytes having TIL-like characteristics), and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved and optionally assessed for phenotype and metabolic parameters as indicators of TIL health.

Patient tumor samples may be obtained using methods known in the art, typically by surgical excision, needle aspiration of biopsy, or other means for obtaining samples containing a mixture of tumor and TIL cells. In general, a tumor sample may be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematopoietic malignancy. The solid tumor may be of any cancer type including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and Neck Squamous Cell Carcinoma (HNSCC)), neuroglioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, suitable TILs are obtained from malignant melanoma tumors, as reports indicate that these tumors have particularly high levels of TILs.

Once obtained, the tumor sample is typically broken into small pieces of between 1mm ³ and about 8mm ³, with about 2 to 3mm ³ being particularly useful, using sharp instrument splitting. TIL was cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., roswell park cancer institute (RPMI) 1640 buffer, 2mM glutamate, 10mcg/mL gentamicin, 30 units/mL dnase, and 1.0mg/mL collagenase), followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by: tumors were placed in enzyme medium and mechanically dissociated for about 1 min, followed by incubation in 5% CO ₂ at 37 ℃ for 30min, followed by repeated mechanical dissociation and incubation cycles under the aforementioned conditions until only small tissue pieces were present. At the end of this process, if the cell suspension contains a large number of erythrocytes or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. patent application publication 2012/0244233 A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in the methods of amplifying TIL or methods of treating cancer in any of the embodiments described herein.

As noted above, in some embodiments, the TIL is derived from a solid tumor. In some embodiments, the solid tumor is not disrupted. In some embodiments, the solid tumor is not disrupted and the enzyme digestion is performed with the intact tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase at 37 ℃ for 1 to 2 hours at 5% CO ₂. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase, and hyaluronidase at 37 ℃ with 5% CO ₂ under rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37 ℃, 5% CO ₂, constant rotation. In some embodiments, the entire tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is 100mg/mL 10X working stock.

In some embodiments, the enzyme mixture comprises dnase. In some embodiments, the working stock of dnase is 10,000iu/mL 10X working stock.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10mg/mL 10X working stock solution.

In general, a cell suspension obtained from a tumor is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population. In certain embodiments, a freshly obtained TIL cell population is exposed to a cell culture medium comprising antigen presenting cells, IL-12 and OKT-3.

In some embodiments, crushing includes physical crushing, including, for example, segmentation and digestion. In some embodiments, the crushing is physical crushing. In some embodiments, the fracture is split. In some embodiments, the disruption is by digestion. In some embodiments, the TIL may be initially cultured from enzymatic tumor digests and tumor fragments obtained from the patient. In some embodiments, the TIL may be initially cultured from enzymatic tumor digests and tumor fragments obtained from the patient.

In some embodiments, when the tumor is a solid tumor, the tumor is physically disrupted after obtaining a tumor sample, e.g., in step a (as provided in particular, e.g., in fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the crushing is performed prior to cryopreservation. In some embodiments, the crushing is performed after cryopreservation. In some embodiments, the disruption is performed after the tumor is obtained and without any cryopreservation. In some embodiments, the disruption step is an in vitro or ex vivo process. In some embodiments, the tumor is disrupted and more than 10, 20, 30, 40 fragments or pieces are placed in each container for initial first expansion. In some embodiments, the tumor is disrupted and 30 or 40 fragments or pieces are placed in each container to initiate the first expansion. In some embodiments, the tumor is disrupted and 40 fragments or pieces are placed in each container for initial first expansion. In some embodiments, the plurality of fragments comprises from about 4 to about 50 fragments, wherein the volume of each fragment is about 27mm ³. In some embodiments, the plurality of fragments comprises from about 30 to about 60 fragments, with a total volume of from about 1300mm ³ to about 1500mm ³. In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350mm ³. In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the TIL is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp segmentation. In some embodiments, the tumor fragments are between about 1mm ³ and 10mm ³. In some embodiments, the tumor fragments are between about 1mm ³ and 8mm ³. In some embodiments, the tumor fragments are about 1mm ³. In some embodiments, the tumor fragments are about 2mm ³. In some embodiments, the tumor fragments are about 3mm ³. In some embodiments, the tumor fragments are about 4mm ³. In some embodiments, the tumor fragments are about 5mm ³. In some embodiments, the tumor fragments are about 6mm ³. In some embodiments, the tumor fragments are about 7mm ³. In some embodiments, the tumor fragments are about 8mm ³. In some embodiments, the tumor fragments are about 9mm ³. In some embodiments, the tumor fragments are about 10mm ³. In some embodiments, the tumor fragments are 1 to 4mm x1 to 4mm. In some embodiments, the tumor fragments are 1mm×1mm. In some embodiments, the tumor fragments are 2mm x 2mm. In some embodiments, the tumor fragments are 3mm×3mm. In some embodiments, the tumor fragments are 4mm x 4mm.

In some embodiments, the tumor is disrupted to minimize the amount of bleeding, necrosis, and/or adipose tissue on each mass. In some embodiments, the tumor is disrupted to minimize the amount of hemorrhagic tissue on each mass. In some embodiments, the tumor is disrupted to minimize the amount of necrotic tissue on each mass. In some embodiments, the tumor is disrupted to minimize the amount of adipose tissue on each mass. In certain embodiments, the tumor disruption step is an in vitro or ex vivo method.

In some embodiments, tumor disruption is performed to maintain tumor internal structure. In some embodiments, tumor disruption is performed without a sawing action using a scalpel. In some embodiments, the TIL is obtained from tumor digests. In some embodiments, the tumor digests are produced by culturing in an enzyme medium (such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10mg/mL gentamicin, 30U/mL dnase, and 1.0mg/mL collagenase), followed by mechanical dissociation (GENTLEMACS of the biotechnology of obumeday, california). After placing the tumor in the enzyme medium, the tumor may be dissociated mechanically for about 1 minute. The solution may then be incubated in 5% CO ₂ at 37 ℃ for 30 minutes, which is then again mechanically disrupted for about 1 minute. After an additional 30 minutes incubation in 5% CO ₂ at 37℃the tumor can be mechanically destroyed a third time for about 1 minute. In some embodiments, if bulk tissue is still present after the third mechanical disruption, 1 or 2 additional mechanical dissociations may be applied to the sample, whether or not incubated in 5% CO ₂ for 30 minutes at 37 ℃. In some embodiments, if at the end of the final culture, the cell suspension contains a large number of erythrocytes or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, the cell suspension prior to initiating the first expansion step is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population.

In some embodiments, the cells may optionally be frozen after sample isolation (e.g., after obtaining a tumor sample and/or after obtaining a cell suspension from a tumor sample), stored frozen prior to entering the expansion described in step B, which is described in further detail below and illustrated in fig. 8 (e.g., fig. 8B in particular).

1. Boll/small biopsy derived TIL

In some embodiments, the TIL is initially obtained from a patient tumor sample obtained by a crude needle biopsy or similar procedure ("primary TIL") and subsequently expanded into a larger population for further manipulation as described herein, optionally cryopreserved and optionally assessed for phenotype and metabolic parameters.

In some embodiments, a patient tumor sample may be obtained using methods known in the art, typically by small biopsy, coarse biopsy, needle aspiration biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In general, a tumor sample may be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematopoietic malignancy. In some embodiments, the sample may be from a plurality of small tumor samples or a biopsy. In some embodiments, the sample may comprise multiple tumor samples from a single tumor of the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three, or four tumors of the same patient. In some embodiments, the sample may comprise multiple tumor samples from multiple tumors of the same patient. The solid tumor may be lung cancer and/or non-small cell lung cancer (NSCLC).

In general, a cell suspension obtained from a tumor core or fragment is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population. In certain embodiments, a freshly obtained TIL cell population is exposed to a cell culture medium comprising antigen presenting cells, IL-2 and OKT-3.

In some embodiments, if the tumor is a metastatic tumor and the primary lesion has been effectively treated/removed in the past, it may be desirable to remove one metastatic lesion. In some embodiments, the minimally invasive method is to remove lymph nodes on the skin lesion or neck or axillary region, if available. In some embodiments, the skin lesion is removed or a small biopsy thereof is removed. In some embodiments, the lymph node or small biopsy thereof is removed. In some embodiments, the tumor is melanoma. In some embodiments, the small biopsy of melanoma comprises a black mole or a portion thereof.

In some embodiments, the small biopsy is a perforated biopsy. In some embodiments, the perforated biopsy is obtained with a circular blade pressed into the skin. In some embodiments, the perforated biopsy is obtained with a circular blade pressed into the skin surrounding the suspicious mole. In some embodiments, a perforated biopsy is obtained with a circular blade pressed into the skin, and a piece of circular skin is removed. In some embodiments, the small biopsy is a perforated biopsy and the tumor of the circular portion is removed.

In some embodiments, the small biopsy is a resected biopsy. In some embodiments, the small biopsy is a resected biopsy and the entire black mole or growths are removed. In some embodiments, the small biopsy is a resected biopsy and the normal appearance skin along with the small edge removes the entire black mole or growths.

In some embodiments, the small biopsy is a cut-out biopsy. In some embodiments, the small biopsy is a cut-out biopsy and only the most irregular portion of the moles or growths are collected. In some embodiments, the small biopsy is a cut-out biopsy, and the cut-out biopsy is used when other techniques cannot be completed, such as when suspicious moles are very large.

In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Generally, bronchoscopy passes a small tool under patient anesthesia through the nose or mouth, down the throat and into the bronchial passages, where the small tool is used to remove some tissue. In some embodiments, percutaneous aspiration of biopsy may be used in cases where a tumor or growth cannot be reached by bronchoscopy. In general, for percutaneous aspiration of living tissue sections, the patient is also under anesthesia and a needle is inserted directly through the skin into the suspicious site to remove a small sample of tissue. In some embodiments, aspiration of a biopsy of body tissue through a brooch may require interventional radiology (e.g., using an X-ray or CT scanning guide needle). In some embodiments, the small biopsy is obtained by needle aspiration of the biopsy. In some embodiments, small biopsy is obtained endoscopically (e.g., endoscopically attached to a lamp and placed transorally in the esophagus). In some embodiments, the small biopsy is obtained surgically.

In some embodiments, the small biopsy is a head and neck biopsy. In some embodiments, the small biopsy is a cut-out biopsy. In some embodiments, the small biopsy is a cut-out biopsy, wherein a small piece of tissue is excised from the region of abnormal appearance. In some embodiments, if the abnormal area is easily accessed, the sample may be collected without hospitalization. In some embodiments, if the tumor is deeper inside the mouth or throat, the biopsy may need to be performed under general anesthesia in the operating room. In some embodiments, the small biopsy is a resected biopsy. In some embodiments, the small biopsy is a resected biopsy, wherein the entire region is removed. In some embodiments, the small biopsy is Fine Needle Aspiration (FNA). In some embodiments, the small biopsy is Fine Needle Aspiration (FNA), in which the cells are extracted (aspirated) from the tumor or tumor mass using a very fine needle attached to a syringe. In some embodiments, the small biopsy is a perforated biopsy. In some embodiments, the small biopsy is a perforated biopsy, wherein a piece of suspicious region is removed using a perforation forceps.

In some embodiments, the small biopsy is a cervical biopsy. In some embodiments, the small biopsy is obtained through a colposcope. Typically, the colposcopic method employs a lamp-attached magnifying instrument (colposcope) attached to a binocular magnifying glass, which is then used to perform a biopsy of a small portion of the cervix. In some embodiments, the small biopsy is a cervical cone resection/cone biopsy. In some embodiments, the small biopsy is a cervical cone resection/cone biopsy, where an outpatient procedure may be required to remove a larger piece of tissue from the cervix. In some embodiments, in addition to facilitating definitive diagnosis, cone-shaped biopsy may also be used as an initial treatment.

The term "solid tumor" refers to an abnormal mass of tissue that does not typically contain cysts or liquid areas. Solid tumors may be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic or cancerous solid tumor. Solid tumor cancers include lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The tissue structure of a solid tumor includes interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells with cancer cells dispersed therein and which can provide a supporting microenvironment.

In some embodiments, the sample from the tumor is obtained in the form of a Fine Needle Aspirate (FNA), a coarse needle biopsy, a small biopsy (including, for example, a punch biopsy). In some embodiments, the sample is first placed in G-REX-10. In some embodiments, when there are 1 or 2 coarse needle biopsy and/or small biopsy samples, the sample is first placed in G-REX-10. In some embodiments, when there are more than 3, 4, 5, 6, 8, 9, or 10 coarse needle biopsy and/or small biopsy samples, the sample is first placed in G-REX-100. In some embodiments, when there are more than 3, 4, 5, 6, 8, 9, or 10 coarse needle biopsy and/or small biopsy samples, the sample is first placed in G-REX-500.

FNA can be obtained from skin tumors, including, for example, melanoma. In some embodiments, the FNA is obtained from a skin tumor, for example, from a patient with metastatic melanoma. In some cases, patients with melanoma have previously undergone surgical treatment.

FNA can be obtained from lung tumors, including, for example, NSCLC. In some embodiments, the FNA is obtained from a lung tumor, e.g., a lung tumor from a non-small cell lung cancer (NSCLC) patient. In some cases, the NSCLC patient has been previously treated by surgery.

TILs described herein may be obtained from FNA samples. In some cases, the FNA sample is obtained or isolated from the patient using a small gauge needle in the range of 18 gauge needle to 25 gauge needle. The small gauge needle may be 18 gauge, 19 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge, 24 gauge, or 25 gauge. In some embodiments, the FNA sample from the patient can contain at least 400,000 TILs, such as 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

In some cases, the TILs described herein are obtained from a coarse needle biopsy sample. In some cases, the coarse needle biopsy sample is obtained or isolated from the patient using a surgical or medical needle in the range of 11 gauge needle to 16 gauge needle. The needle may be 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge, or 16 gauge. In some embodiments, a coarse needle biopsy sample from a patient may contain at least 400,000 TILs, such as 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

In some embodiments, the TIL is not obtained from tumor digests. In some embodiments, the solid tumor core is not disrupted.

In some embodiments, the TIL is obtained from tumor digests. In some embodiments, the tumor digests are produced by incubation in an enzyme medium (such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10mg/mL gentamicin, 30U/mL dnase, and 1.0mg/mL collagenase), followed by mechanical dissociation (GENTLEMACS of the biotechnology of obumeday, california). After placing the tumor in the enzyme medium, the tumor may be dissociated mechanically for about 1 minute. The solution may then be incubated in 5% CO ₂ at 37 ℃ for 30 minutes, which is then again mechanically disrupted for about 1 minute. After an additional 30 minutes incubation in 5% CO ₂ at 37℃the tumor can be mechanically destroyed a third time for about 1 minute. In some embodiments, if bulk tissue is still present after the third mechanical disruption, 1 or 2 additional mechanical dissociations may be applied to the sample, whether or not incubated in 5% CO ₂ for 30 minutes at 37 ℃. In some embodiments, if at the end of the final incubation the cell suspension contains a large number of erythrocytes or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, obtaining the first TIL population comprises a multi-foci sampling method.

The tumor dissociating enzyme mixture may comprise one or more dissociating (digesting) enzymes, such as, but not limited to, collagenase (including any mixed or type of collagenase), accutase ^TM、Accumax^TM, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, XIV-type protease (chain protease), deoxyribonuclease I (dnase), trypsin inhibitors, any other dissociating or proteolytic enzyme, and any combination thereof.

In some embodiments, the dissociating enzyme is reconstituted by a freeze-drying enzyme. In some embodiments, the lyophilized enzyme is reconstituted in an amount of a sterile buffer, such as Hank's Balanced Salt Solution (HBSS).

In some cases, the collagenase (e.g., no animal-derived collagenase type 1) is reconstituted in 10mL of sterile HBSS or another buffer. The concentration of lyophilized stock enzyme may be 2892PZ U per vial. In some embodiments, the collagenase is reconstituted in 5mL to 15mL buffer. In some embodiments, the collagenase stock solution ranges from about 100PZ U/mL to about 400PZ U/mL after reconstitution, for example, from about 100PZ U/mL to about 400PZ U/mL, from about 100PZ U/mL to about 350PZ U/mL, from about 100PZ U/mL to about 300PZ U/mL, from about 150PZ U/mL to about 400PZ U/mL, from about 100PZ U/mL, about 150PZ U/mL, about 200PZ U/mL, about 210PZ U/mL, about 220PZ U/mL, about 230PZ U/mL, about 240PZ U/mL, about 250PZ U/mL, about 260PZ U/mL, about 270PZ U/mL, about 280PZ U/mL, about 289.2PZ U/mL, about 300PZ U/mL, about 350PZ U/mL, or about 400PZ U/mL.

In some embodiments, the enzyme stock solution will change, thus verifying the concentration of the lyophilized stock solution and modifying the final amount of enzyme added to the digestion mixture accordingly.

2. Pleural effusion T cells and TIL

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion according to the processes described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of T cells or TILs for expansion according to the processes described herein is a pleural effusion-derived sample. See, for example, the method described in U.S. patent publication US2014/0295426, which is incorporated by reference herein in its entirety for all purposes.

In some embodiments, any pleural or pleural effusion suspected of and/or containing TIL may be employed. Such samples may be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample may be a secondary metastatic cancer cell derived from another organ (e.g., breast, ovary, colon, or prostate). In some embodiments, the sample used in the amplification methods described herein is pleural effusion. In some embodiments, the sample used in the amplification methods described herein is pleural effusion. Other biological samples may include other slurries containing TIL, including, for example, ascites fluid from the abdomen or pancreatic cyst fluid. Ascites fluid and pleural fluid involve very similar chemical systems; both the abdomen and the lung have mesothelial cell lines and fluid forms in the thorax and abdominal cavities in the same malignant event, in some embodiments such fluids contain TIL. In some embodiments of the present exemplary pleural fluid, the same procedure may be performed using ascites or other cyst fluid containing TIL to achieve similar results.

In some embodiments, pleural fluid is removed directly from the patient in untreated form. In some embodiments, prior to the contacting step, untreated pleural fluid is placed in a standard blood collection tube (e.g., EDTA or heparin tube). In some embodiments, the untreated pleural fluid is placed in a standard prior to the contacting stepIn a tube (Veridex). In some embodiments, the sample is placed in the CellSave tube immediately after collection from the patient to avoid a decrease in the number of active TILs. If left in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4 ℃. In some embodiments, the sample is placed in a suitable collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in a suitable collection tube 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4 ℃.

In other embodiments, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, pre-treatment of pleural fluid is preferred in cases where such fluid must be cryopreserved for shipment to a laboratory where the method is performed or for subsequent analysis (e.g., 24 to 48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is removed from the subject and re-suspending the centrifugate or precipitate in buffer. In some embodiments, the pleural fluid sample is centrifuged and resuspended multiple times and then cryopreserved for shipment or later analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated by using a filtration method prior to further processing steps. In some embodiments, the pleural fluid sample used in the contacting step is prepared by filtering the fluid through a filter containing a known and substantially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the pores in the membrane may be at least 4 μm in diameter. In other embodiments, the pore size may be above 5 μΜ, and in other embodiments, may be any of 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ or 10 μΜ. After filtration, the cells (including TIL) retained by the membrane may be washed from the membrane into a suitable physiologically acceptable buffer. Cells concentrated in this way (including TIL) can then be used in the contacting step of the method.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysing agent that differentially lyses the non-nucleated red blood cells present in the sample. In some embodiments, where the pleural fluid contains a large number of RBCs, this step is performed prior to further processing steps. Suitable lysing reagents include a single lysing reagent or a lysing reagent and a quenching reagent, or a lysing reagent, a quenching reagent, and an immobilization reagent. Suitable lysis systems are commercially available, including the BD PHARM LYSE ^TM system (bi healthcare). Other lysis systems include VERSALYSE ^TM systems, FACSlyse ^TM systems (Bidi medical Co.), immunoprep ^TM systems or Erythrolyse II systems (Beckmann Coulter) or ammonium chloride systems. In some embodiments, the lysing agent may vary with the primary requirements, which are efficient lysis of erythrocytes and conservation of TIL and phenotypic characteristics of TIL in pleural fluid. In addition to using a single reagent for cleavage, a cleavage system suitable for use in the methods described herein may include a second reagent, such as a Stabilyse ^TM reagent (Beckmann Coulter) that quenches or delays the action of the cleavage reagent during the remaining steps of the method. Depending on the choice of cleavage reagent or the preferred implementation of the method, conventional immobilization reagents may also be used.

3. Method for amplifying Peripheral Blood Lymphocytes (PBLs) derived from peripheral blood

PBL method 1. In some embodiments of the invention, the PBLs are amplified using the methods described herein. In some embodiments of the invention, the method comprises obtaining a PBMC sample from whole blood. In some embodiments, the method comprises enriching T cells by using negative selection of non-cd19+ fractions to isolate pure T cells from PBMCs. In some embodiments, the method comprises enriching T cells by using bead-based negative selection of non-cd19+ fractions to isolate pure T cells from PBMCs.

In some embodiments of the invention, PBL method 1 proceeds as follows: on day 0, cryopreserved PBMC samples were thawed and PBMC numbers were counted. T cells were isolated using the human pan T cell isolation kit with LS column (meitian gentle biotechnology).

PBL method 2. In some embodiments of the invention, the PBLs are amplified using PBL method 2, which includes obtaining PBMC samples from whole blood. T cells from PBMCs were enriched by incubating PBMCs at 37 ℃ for at least three hours, followed by isolation of non-adherent cells.

In some embodiments of the invention, PBL method 2 proceeds as follows: on day 0, the cryopreserved PMBC samples were thawed, PBMC cells were seeded at 6 million cells per well in 6-well plates in CM-2 medium and incubated for 3 hours at 37 ℃. After 3 hours, non-adherent cells (which are PBLs) were removed and counted.

PBL method 3. In some embodiments of the invention, the PBLs are amplified using PBL method 3, which includes obtaining PBMC samples from peripheral blood. B cells were isolated using cd19+ selection and T cells were selected using non-cd19+ fractions of negative selection PBMC samples.

In some embodiments of the invention, PBL method 3 proceeds as follows: on day 0, cryopreserved PBMCs from peripheral blood were thawed and counted. Cd19+ B cells were sorted using the human CD19Multisort kit (meitian gentle biotechnology). In the non-cd19+ cell fraction, T cells were purified using a human pan T cell isolation kit and LS column (meitian gentle biotechnology).

In some embodiments of the invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify non-adherent cells. In some embodiments of the invention, the incubation time is about 3 hours. In some embodiments of the invention, the temperature is about 37 ℃. Non-adherent cells are then expanded using the procedure described above.

In some embodiments of the invention, on day 0, cells are selected for cd19+ and sorted accordingly. In some embodiments of the invention, the selection is performed using antibody binding beads. In some embodiments of the invention, the pure T cells are isolated from PBMCs on day 0.

In some embodiments of the invention, for patients not pretreated with ibutenib or other ITK inhibitor, 10 to 15mL of the buffy coat will produce about 5 x 10 ⁹ PBMCs, which in turn will produce about 5.5 x 10 ⁷ PBLs.

In some embodiments of the invention, the amplification process will yield about 20 x 10 ⁹ PBLs for patients pretreated with ibutinib or other ITK inhibitor. In some embodiments of the invention, 40.3X10 ⁶ PBMC will produce about 4.7X10 ⁵ PBLs.

In some embodiments, the PBL is prepared using the method described in U.S. patent application publication No. US2020/0347350 A1, the disclosure of which is incorporated herein by reference.

4. Method for amplifying bone Marrow Infiltrating Lymphocytes (MILs) from bone marrow derived PBMCs

MILs method 3. In some embodiments of the invention, the method comprises obtaining PBMCs from bone marrow. On day 0, PBMCs were selected for cd3+/cd33+/cd20+/cd14+, sorted, non-cd3+/cd33+/cd20+/cd14+ cell fractions sonicated, and a portion of the sonicated cell fraction was added back to the selected cell fraction.

In some embodiments of the invention, MILs method 3 proceeds as follows: on day 0, cryopreserved PBMC samples were thawed and PBMC numbers were counted. Cells were stained with CD3, CD33, CD20 and CD14 antibodies and sorted using an S3e cell sorter (Bio-Rad). Cells were sorted into two fractions: immune cell fraction (MILs fraction) (cd3+cd33+cd20+cd14+) and AML blast fraction (non-cd3+cd33+cd20+cd14+).

In some embodiments of the invention, the PBMCs are obtained from bone marrow. In some embodiments, the PBMCs are obtained from bone marrow by apheresis, aspiration, needle aspiration of a biopsy or other similar means known in the art. In some embodiments, the PBMCs are fresh. In other embodiments, the PBMCs are cryopreserved.

In some embodiments of the invention, MILs are expanded from 10 to 50mL bone marrow aspirate. In some embodiments of the invention, 10mL bone marrow aspirate is obtained from a patient. In other embodiments, 20mL bone marrow aspirate is obtained from a patient. In other embodiments, 30mL bone marrow aspirate is obtained from a patient. In other embodiments, 40mL bone marrow aspirate is obtained from a patient. In other embodiments, 50mL bone marrow aspirate is obtained from a patient.

In some embodiments of the invention, the number of PBMCs generated from about 10 to 50mL of bone marrow aspirate is about 5 x 10 ⁷ to about 10 x 10 ⁷ PBMCs. In other embodiments, the number of PMBCs produced is about 7 x 10 ⁷ PBMCs.

In some embodiments of the invention, about 5×10 ⁷ to about 10×10 ⁷ PBMCs produce about 0.5×10 ⁶ to about 1.5×10 ⁶ MILs. In some embodiments of the invention, about 1×10 ⁶ MILs are produced.

In some embodiments of the invention, 12×10 ⁶ PBMCs derived from bone marrow aspirate produce about 1.4×10 ⁵ MILs.

In any of the foregoing embodiments, the PBMCs may be derived from whole blood samples, bone marrow, obtained by apheresis, derived from white film, or obtained from any other method known in the art for obtaining PBMCs.

In some embodiments, MILs are prepared using the methods described in U.S. patent application publication No. US2020/0347350 A1, the disclosure of which is incorporated herein by reference.

B. And (B) step (B): initiation of first amplification

In some embodiments, the methods of the invention provide a younger TIL that may provide additional therapeutic benefit compared to an older TIL (i.e., a TIL that has been replicated more times further prior to administration to a subject/patient). The characteristics of young TILs have been described in the literature, for example Donia et al, journal of scandinavia immunology (scand.j.immunol.) 2012,75,157-167; dudley et al, clinical cancer research (Clin. Cancer Res.) 2010,16,6122-6131; huang et al, journal of immunology 2005,28,258-267; besser et al, clinical cancer research 2013,19, OF1-OF9; besser et al, journal of immunology 2009,32,415-423; robbins et al, journal of immunology 2004,173,7125-7130; shen et al, journal of immunology 2007,30,123-129; zhou et al, journal of immunology 2005,28,53-62; and Tran et al, journal of immunology 2008,31,742-751, each of which is incorporated herein by reference.

Following the segmentation or digestion of tumor fragments and/or tumor fragments, e.g., as described in step a of fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C), the resulting cells are cultured in serum containing IL-2, OKT-3 and feeder cells (e.g., antigen presenting feeder cells) under conditions that favor TIL but disfavor tumor and other cell growth. In some embodiments, IL-2, OKT-3 and feeder cells are added at the beginning of the culture (e.g., on day 0) along with tumor digests and/or tumor fragments. In some embodiments, tumor digests and/or tumor fragments are incubated in the container with up to 60 fragments per container and 6000IU/mL IL-2. In some embodiments, this primary cell population is cultured for a period of typically 1 to 8 days, resulting in a subject TIL population of typically about 1 x 10 ⁸ subject TIL cells. In some embodiments, this primary cell population is cultured for a period of typically 1 to 7 days, resulting in a subject TIL population of typically about 1 x 10 ⁸ subject TIL cells. In some embodiments, the initial first expansion is performed for a period of 1 to 8 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, the initial first expansion is performed for a period of 1 to 7 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, this initial first expansion is performed for a period of 5 to 8 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, this initial first expansion is performed for a period of 5 to 7 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, this initial first expansion is performed for a period of about 6 to 8 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, this initial first expansion is performed for a period of about 6 to 7 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, this initial first expansion is performed for a period of about 7 to 8 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, this initial first expansion is performed for a period of about 7 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells. In some embodiments, this initial first expansion is performed for a period of about 8 days, resulting in a population of host TILs of typically about 1 x 10 ⁸ host TIL cells.

In some embodiments, the amplification of TIL may be performed using an initial first amplification step (e.g., those described in step B of fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D) as described below and herein, which may include a process called pre-REP or initial REP that contains feeder cells since day 0 and/or since culture), followed by a rapid second amplification (step D, including a process called a rapid amplification protocol (REP) step) as described below and herein), followed by an optional cryopreservation, followed by a second step D (including a process called a re-stimulus REP step) as described below and herein. TIL obtained from this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some embodiments, the tumor fragments are between about 1mm ³ and 10mm ³.

In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for medium). In some embodiments, the CM of step B consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25mM Hepes, and 10mg/mL gentamicin.

In some embodiments, there are less than or equal to 240 tumor fragments. In some embodiments, less than or equal to 240 tumor fragments are placed in less than or equal to 4 containers. In some embodiments, the container is GREX MCS flask. In some embodiments, less than or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each container contains less than or equal to 500mL of medium per container. In some embodiments, the medium comprises IL-2. In some embodiments, the medium contains 6000IU/mL IL-2. In some embodiments, the medium comprises antigen presenting feeder cells (also referred to herein as "antigen presenting cells"). In some embodiments, the medium comprises 2.5X10 ⁸ antigen presenting feeder cells/container. In some embodiments, the medium comprises OKT-3. In some embodiments, the medium comprises 30ng/mL OKT-3/vessel. In some embodiments, the container is GREX MCS flask. In some embodiments, the medium contains 6000IU/mL IL-2, 30ng OKT-3 and 2.5X10 ⁸ antigen presenting feeder cells. In some embodiments, the medium contains 6000IU/mL IL-2, 30ng/mL OKT-3 and 2.5X10 ⁸ antigen presenting feeder cells/container.

After preparation of tumor fragments, the resulting cells (i.e., fragments that are primary cell populations) are cultured in a medium containing IL-2, antigen presenting feeder cells, and OKT-3 under conditions that favor TIL but disfavor tumor and other cell growth, which allows TIL initiation and acceleration from day 0 culture. In some embodiments, tumor digests and/or tumor fragments are incubated with 6000IU/mL IL-2, antigen presenting feeder cells, and OKT-3. This primary cell population is cultured for a period of days, typically 1 to 8 days, resulting in a population of bulk TIL, typically about 1 x 10 ⁸ bulk TIL cells. In some embodiments, the growth medium during initiation of the first amplification comprises IL-2 or a variant thereof, as well as antigen presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for a period of days, typically 1 to 7 days, resulting in a subject TIL population of typically about 1 x 10 ⁸ subject TIL cells. In some embodiments, the growth medium during initiation of the first amplification comprises IL-2 or a variant thereof, as well as antigen presenting feeder cells and OKT-3. In some embodiments, IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, a 1mg vial of IL-2 stock solution has a specific activity of 20 to 30X 10 ⁶ IU/mg. In some embodiments, a 1mg vial of IL-2 stock has a specific activity of 20X 10 ⁶ IU/mg. In some embodiments, a 1mg vial of IL-2 stock has a specific activity of 25X 10 ⁶ IU/mg. In some embodiments, a 1mg vial of IL-2 stock has a specific activity of 30X 10 ⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4 to 8X 10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5 to 7X 10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6X 10 ⁶ IU/mg IL-2. In some embodiments, IL-2 stock solution is prepared as described in example C. In some embodiments, the initial first amplification medium comprises about 10,000IU/mL IL-2, about 9,000IU/mL IL-2, about 8,000IU/mL IL-2, about 7,000IU/mL IL-2, about 6000IU/mL IL-2, or about 5,000IU/mL IL-2. In some embodiments, the starting first amplification medium comprises from about 9,000IU/mL IL-2 to about 5,000IU/mL IL-2. In some embodiments, the starting first amplification medium comprises from about 8,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the starting first amplification medium comprises from about 7,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the starting first amplification medium comprises about 6,000IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the starting first expanded cell culture medium comprises about 3000IU/mL IL-2. In some embodiments, the starting first expanded cell culture medium further comprises IL-2. In some embodiments, the starting first expanded cell culture medium comprises about 3000IU/mL IL-2. In some embodiments, the starting first expanded cell culture medium comprises about 1000IU/mL, about 1500IU/mL, about 2000IU/mL, about 2500IU/mL, about 3000IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, or about 8000IU/mL IL-2. In some embodiments, the starting first expanded cell culture medium comprises 1000 to 2000IU/mL, 2000 to 3000IU/mL, 3000 to 4000IU/mL, 4000 to 5000IU/mL, 5000 to 6000IU/mL, 6000 to 7000IU/mL, 7000 to 8000IU/mL, or about 8000IU/mL IL-2.

In some embodiments, the initial first amplification medium comprises about 500IU/mL IL-15, about 400IU/mL IL-15, about 300IU/mL IL-15, about 200IU/mL IL-15, about 180IU/mL IL-15, about 160IU/mL IL-15, about 140IU/mL IL-15, about 120IU/mL IL-15, or about 100IU/mL IL-15. In some embodiments, the starting first amplification medium comprises about 500IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the starting first amplification medium comprises about 400IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the starting first amplification medium comprises about 300IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the initial first amplification medium comprises about 200IU/mL IL-15. In some embodiments, the starting first expanded cell culture medium comprises about 180IU/mL IL-15. In some embodiments, the starting first expanded cell culture medium further comprises IL-15. In some embodiments, the starting first expanded cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the initial first amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mL IL-21. In some embodiments, the starting first amplification medium comprises about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the starting first amplification medium comprises from about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the starting first amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the starting first amplification medium comprises from about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the starting first amplification medium comprises from about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the initial first amplification medium comprises about 2IU/mL IL-21. In some embodiments, the starting first expanded cell culture medium comprises about 1IU/mL IL-21. In some embodiments, the starting first expanded cell culture medium comprises about 0.5IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the starting first expanded cell culture medium comprises about 1IU/mL IL-21.

In some embodiments, the starting first expanded cell culture medium comprises an OKT-3 antibody. In some embodiments, the starting first expanded cell culture medium comprises about 30ng/mL OKT-3 antibody. In some embodiments, the starting first expanded cell culture medium comprises about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1 μg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises OKT-3 antibodies between 0.1ng/mL and 1ng/mL, between 1ng/mL and 5ng/mL, between 5ng/mL and 10ng/mL, between 10ng/mL and 20ng/mL, between 20ng/mL and 30ng/mL, between 30ng/mL and 40ng/mL, between 40ng/mL and 50ng/mL, and between 50ng/mL and 100 ng/mL. In some embodiments, the cell culture medium comprises 15ng/mL to 30ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises 30ng/mL OKT-3 antibody. In some embodiments, the OKT-3 antibody is Moromolizumab. See, e.g., table 1.

In some embodiments, the starting first expanded cell culture medium comprises more than one TNFRSF agonist in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: wu Ruilu mab, wu Tumu mab, EU-101, fusion proteins, fragments, derivatives, variants, biological analogs, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 μg/mL and 100 μg/mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 μg/mL and 40 μg/mL.

In some embodiments, the starting first expanded cell culture medium further comprises IL-2 at an initial concentration of about 3000IU/mL and OKT-3 antibody at an initial concentration of about 30ng/mL in addition to the one or more TNFRSF agonists, wherein the one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, the starting first expanded cell culture medium further comprises IL-2 at an initial concentration of about 6000IU/mL and OKT-3 antibody at an initial concentration of about 30ng/mL in addition to the one or more TNFRSF agonists, wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the starting first amplification medium is referred to as "CM" (abbreviation for medium). In some embodiments, it is referred to as CM1 (medium 1). In some embodiments, CM consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25mM Hepes, and 10mg/mL gentamicin. In some embodiments, the CM is CM1 described in the examples. In some embodiments, initiating the first expansion is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial first expansion medium or primary cell culture medium or first cell culture medium comprises IL-2, OKT-3, and antigen presenting feeder cells (also referred to herein as feeder cells).

In some embodiments, the serum-free or defined medium is CTS ^TMOpTmizer^TM T cell expansion SFM (sameir feishier technology). Any CTS ^TMOpTmizer^TM formulation may be used in the present invention. CTS ^TMOpTmizer^TM T cell expansion SFM is a combination of 1L CTS ^TMOpTmizer^TM T cell expansion basal medium with 26mL CTS ^TMOpTmizer^TM T cell expansion supplement, which is mixed together prior to use. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sameir feishier technology). In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), the final concentration of 2-mercaptoethanol in the medium is 55 μm. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology) and 55 μm 2-mercaptoethanol.

In some embodiments, the defined medium is CTS ^TMOpTmizer^TM T cell expansion SFM (sammer feishier technology). Any CTS ^TMOpTmizer^TM formulation may be used in the present invention. CTS ^TMOpTmizer^TM T cell expansion SFM is a combination of 1L CTS ^TMOpTmizer^TM T cell expansion basal medium with 26mL CTS ^TMOpTmizer^TM T cell expansion supplement, which is mixed together prior to use. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), the final concentration of 2-mercaptoethanol in the medium is 55 μm.

In some embodiments, defined media as described in Smith et al, clinical transformation immunology, 4 (1), 2015 (doi: 10.1038/cti.2014.31) may be used in the present invention. Briefly, RPMI or CTS ^TMOpTmizer^TM was used as basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS ^TM immune cell serum replacement.

In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 1 to 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 2 to 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 3 to 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 4 to 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 5 to 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 6 to 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 1 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 7 to 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 8 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 1 to 7 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 2 to 7 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 3 to 7 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 4 to 7 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8B and/or fig. 8C), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 5 to 7 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 6 to 7 days, as described in the examples and illustrations. In some embodiments, the first amplification process (including, for example, those described in step B of fig. 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D), which may include those sometimes referred to as pre-REP or initial REP) is initiated for 7 days, as described in the examples and illustrations.

In some embodiments, the initial first TIL amplification may be performed 1 day to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 1 day to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 2 days to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 2 days to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 3 days to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 3 days to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 4 days to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 4 days to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 5 days to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 5 days to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed from 6 days to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed from 6 days to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 7 to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the initial first TIL amplification may be performed 7 days after disruption is performed and/or after the first initial amplification step is initiated.

In some embodiments, the initial first amplification of TIL may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days. In some embodiments, the first TIL amplification may be performed for 1 day to 8 days. In some embodiments, the first TIL amplification may be performed for 1 day to 7 days. In some embodiments, the first TIL amplification may be performed for 2 days to 8 days. In some embodiments, the first TIL amplification may be performed for 2 days to 7 days. In some embodiments, the first TIL amplification may be performed for 3 days to 8 days. In some embodiments, the first TIL amplification may be performed for 3 days to 7 days. In some embodiments, the first TIL amplification may be performed for 4 days to 8 days. In some embodiments, the first TIL amplification may be performed for 4 days to 7 days. In some embodiments, the first TIL amplification may be performed for 5 days to 8 days. In some embodiments, the first TIL amplification may be performed for 5 days to 7 days. In some embodiments, the first TIL amplification may be performed for 6 days to 8 days. In some embodiments, the first TIL amplification may be performed for 6 days to 7 days. In some embodiments, the first TIL amplification may be performed for 7 days to 8 days. In some embodiments, the first TIL amplification may be performed for 8 days. In some embodiments, the first TIL amplification may be performed for 7 days.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is employed as the combination during initiation of the first amplification. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included during initiation of the first amplification, including, for example, during a process according to FIG. 8 (particularly, e.g., FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D) and step B described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is employed as the combination during initiation of the first amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG. 8 (e.g., FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D, in particular) and step B as described herein.

In some embodiments, initiating the first amplification (e.g., according to step B) of fig. 8 (e.g., particularly fig. 8A and/or 8B and/or 8C and/or 8D) is performed in a closed system bioreactor. In some embodiments, the TIL amplification as described herein is performed using a closed system. In some embodiments, a bioreactor is employed. In some embodiments, a bioreactor is employed as the vessel. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

1. Feeder cells and antigen presenting cells

In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or 8B and/or 8C and/or 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of the TIL amplification, but rather is added during the initial first amplification. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or 8B and/or 8C and/or 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during days 4 to 8 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or 8B and/or 8C and/or 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during days 4 to 7 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during days 5 to 8 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or 8B and/or 8C and/or 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during days 5 to 7 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during days 6 to 8 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or 8B and/or 8C and/or 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during days 6 to 7 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during day 7 or 8 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly such as fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during day 7 of the initial first amplification period. In some embodiments, the initial first amplification procedure described herein (e.g., including amplifications such as those described in step B of fig. 8 (particularly, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D) and those referred to as pre-REP or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but is added at any time during day 8 of the initial first amplification period.

In some embodiments, initiating a first amplification procedure described herein (e.g., including amplification such as those described in step B of fig. 8 (particularly e.g., fig. 8B) and those referred to as pre-REP or initial REP) requires feeder cells (also referred to herein as "antigen presenting cells") at the beginning and during initiation of TIL amplification. In many embodiments, the feeder cells are obtained from Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units of allogeneic healthy blood donors. PBMC are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, 2.5X10 ⁸ feeder cells are used during the initial first expansion. In some embodiments, 2.5X10 ⁸ feeder cells per container are used during the initial first expansion. In some embodiments, every GREX-10.5X10 ⁸ feeder cells are used during the initial first expansion. In some embodiments, every GREX-100.5X10 ⁸ feeder cells are used during the initial first expansion.

In some embodiments, PBMCs are considered replication incompetent and acceptable for the TIL expansion procedure described herein if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of initial first expansion.

In some embodiments, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedure described herein if the total number of living cells cultured in the presence of OKT3 and IL-2 on day 7 is not increased compared to the initial number of living cells placed in culture on day 0 of initial first expansion. In some embodiments, PBMC are cultured in the presence of 30ng/mL OKT3 antibody and 3000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 30ng/mL OKT3 antibody and 6000IU/mL IL-2.

In some embodiments, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedure described herein if the total number of living cells cultured in the presence of OKT3 and IL-2 on day 7 is not increased compared to the initial number of living cells placed in culture on day 0 of initial first expansion. In some embodiments, PBMC are cultured in the presence of 5 to 60ng/mL OKT3 antibody and 1000 to 6000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 10 to 50ng/mL OKT3 antibody and 2000 to 5000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 20 to 40ng/mL OKT3 antibody and 2000 to 4000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 25 to 35ng/mL OKT3 antibody and 2500 to 3500IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 30ng/mL OKT3 antibody and 6000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 15ng/mL OKT3 antibody and 3000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 15ng/mL OKT3 antibody and 6000IU/mL IL-2.

In some embodiments, the initial first expansion procedure described herein requires a ratio of about 2.5X10 ⁸ feeder cells to about 100X 10 ⁶ TILs. In other embodiments, the initial first expansion procedure described herein requires a ratio of about 2.5X10 ⁸ feeder cells to about 50X 10 ⁶ TILs. In other embodiments, about 2.5X10 ⁸ feeder cells and about 25X 10 ⁶ TILs are required to initiate the first expansion described herein. In other embodiments, about 2.5X10 ⁸ feeder cells are required to initiate the first expansion described herein. In other embodiments, the number of feeder cells required to initiate the first expansion is one-fourth, one-third, five-twelve or one-half of the number of feeder cells used for the rapid second expansion.

In some embodiments, the medium in which the first amplification is initiated comprises IL-2. In some embodiments, the medium in which the first amplification is initiated comprises 6000IU/mL IL-2. In some embodiments, the medium in which the first expansion is initiated comprises antigen presenting feeder cells. In some embodiments, the medium in which the first expansion is initiated comprises 2.5X10 ⁸ antigen presenting feeder cells per container. In some embodiments, the medium in which the first amplification is initiated comprises OKT-3. In some embodiments, the medium comprises 30ng OKT-3 per container. In some embodiments, the container is GREX MCS flask. In some embodiments, the medium contains 6000IU/mL IL-2, 30ng/mL OKT-3 and 2.5X10 ⁸ antigen presenting feeder cells. In some embodiments, the medium contains 6000IU/mL IL-2, 30ng/mL OKT-3, and 2.5X10 ⁸ antigen presenting feeder cells per container. In some embodiments, the medium comprises 500mL of medium and 15 μg OKT-3 per 2.5X10 ⁸ antigen presenting feeder cells per container. In some embodiments, the medium comprises 500mL of medium and 15 μ gOKT-3 per vessel. In some embodiments, the container is GREX MCS flask. In some embodiments, the medium contains 500mL medium, 6000IU/mL IL-2, 30ng/mLOKT-3 and 2.5X10 ⁸ antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium per container, 6000IU/mL IL-2, 15 μg OKT-3, and 2.5X10 ⁸ antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium and 15 μg OKT-3 per 2.5X10 ⁸ antigen presenting feeder cells per container.

In some embodiments, initiating the first amplification procedure described herein requires an excess of feeder cells over TIL during the second amplification. In many embodiments, the feeder cells are obtained from Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units of allogeneic healthy blood donors. PBMC are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aapcs) are used in place of PBMCs.

In some embodiments, artificial antigen presenting cells are used in place of or in combination with PBMCs in initiating the first expansion.

2. Cytokines and other additives

Alternatively, it is also possible to use cytokines in combination with the following for initial first amplification of TIL: a combination of two or more of IL-2, IL-15 and IL-21 as described in U.S. patent application publication No. US2017/0107490 A1, the disclosures of which are incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, the latter having particular utility in many embodiments. The use of combinations of cytokines is particularly advantageous for the production of lymphocytes, in particular the T cells described therein. See, e.g., table 2.

In some embodiments, step B may also comprise adding OKT-3 antibody or moromiab to the medium, as described elsewhere herein. In some embodiments, step B may also include adding a 4-1BB agonist to the medium, as described elsewhere herein. In some embodiments, step B may also comprise adding an OX-40 agonist to the culture medium, as described elsewhere herein. Furthermore, additives such as peroxisome proliferator activated receptor gamma coactivator I-alpha agonists, including proliferator activated receptor (PPAR) -gamma agonists, such as thiazolidinedione compounds, may be used in the medium during step B, as described in US patent application publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Step C: initiating a transition from a first amplification to a rapid second amplification

In some cases, a population of TILs obtained from a subject that initiates a first amplification (which may include an amplification sometimes referred to as pre-REP), including, for example, a population of TILs obtained from step B, e.g., as indicated in fig. 8 (particularly, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D), may be subjected to a rapid second amplification (which may include an amplification sometimes referred to as a rapid amplification protocol (REP)), and then cryopreserved as described below. Similarly, where a genetically modified TIL is to be used in therapy, the amplified TIL population from the initial first amplification or the amplified TIL population from the rapid second amplification may be genetically modified for appropriate treatment prior to the amplification step or after the initial first amplification and prior to the rapid second amplification.

In some embodiments, TIL obtained from initiating the first amplification (e.g., step B indicated in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is stored until phenotyping is determined for selection. In some embodiments, TIL obtained from initiating the first amplification (e.g., step B indicated in fig. 8 (particularly e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) is not stored and is directly subjected to the rapid second amplification. In some embodiments, TIL obtained from initiating the first amplification is not cryopreserved after initiating the first amplification and before the rapid second amplification. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days after tumor disruption is performed and/or after the initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed about 3 days to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed about 3 days to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 4 days to 7 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 4 days to 8 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 5 days to 7 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 5 days to 8 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 6 days to 7 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 6 days to 8 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed about 7 days to 8 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 8 days after disruption is performed and/or after the first initial amplification step is initiated.

In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 1 day to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 1 day to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed 2 to 7 days after the disruption is performed and/or after the initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification is performed 2 to 8 days after the disruption is performed and/or after the initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification is performed 3 to 7 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the second amplification is performed 3 to 8 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 4 to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 4 to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 5 to 7 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 5 to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 6 to 7 days after the disruption is performed and/or after the first initial amplification step is initiated, in some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 6 to 8 days after the disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 7 to 8 days after disruption is performed and/or after the first initial amplification step is initiated. In some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 7 days after the disruption is performed and/or after the first initial amplification step is initiated, in some embodiments, the transition from initiating the first amplification to the rapid second amplification is performed 8 days after the disruption is performed and/or after the first initial amplification step is initiated.

In some embodiments, TIL is not stored after the primary first amplification (PRIMARY FIRST expansion) and prior to the rapid second amplification, and TIL is directly subjected to the rapid second amplification (e.g., in some embodiments, is not stored during the transition of step B to step D as shown in fig. 8 (e.g., particularly fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D)). In some embodiments, the transition is performed in a closed system as described herein. In some embodiments, TILs from the initial first amplification (second population of TILs) are directly subjected to the rapid second amplification without a transition phase.

In some embodiments, the transition from the initiation of the first amplification to the rapid second amplification (e.g., according to step C) of fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) is performed in a closed system bioreactor. In some embodiments, the TIL amplification as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, a single bioreactor is employed, for example GREX-10 or GREX-100. In some embodiments, the closed system bioreactor is a single bioreactor. In some embodiments, the transition from initiating the first amplification to the rapid second amplification involves a longitudinal expansion in the scale of the container size. In some embodiments, the initial first amplification is performed in a smaller vessel than the rapid second amplification. In some embodiments, the initial first amplification is performed in GREX-100 and the rapid second amplification is performed in GREX-500.

D. Step D: rapid second amplification

In some embodiments, the TIL cell population is further expanded in number after the collection and initiation of the first expansion (step a and step B) and the transition referred to as step C as indicated in fig. 8 (in particular, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). This further amplification is referred to herein as a rapid second amplification or rapid amplification, which may include an amplification process commonly referred to in the art as a rapid amplification process (rapid amplification protocol or REP); and as indicated in step D of fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). Rapid second expansion is typically accomplished in a gas-permeable vessel using a medium comprising a plurality of components, including feeder cells, a cytokine source, and anti-CD 3 antibodies. In some embodiments, the TIL is transferred to the larger volume container 1 day, 2 days, 3 days, or 4 days after initiation of the rapid second amplification (i.e., on day 8, 9, 10, or 11 of the overall Gen3 process).

In some embodiments, the rapid second amplification of TIL (which may include amplification sometimes referred to as REP; and the process as indicated in step D of FIG. 8 (particularly, e.g., FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D)) may be performed using any TIL flask or vessel known to those of skill in the art. In some embodiments, the second TIL amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 1 day to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 2 days to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 2 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 3 days to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 3 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 4 days to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 4 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 5 days to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 5 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 6 days to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 6 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 7 days to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 7 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 8 days to about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 8 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed from about 9 days to about 10 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 1 day after the onset of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 2 days after the rapid second amplification begins. In some embodiments, the second TIL amplification may be performed about 3 days after the rapid second amplification begins. In some embodiments, the second TIL amplification may be performed about 4 days after the rapid second amplification begins. In some embodiments, the second TIL amplification may be performed about 5 days after the rapid second amplification begins. In some embodiments, the second TIL amplification may be performed about 6 days after the rapid second amplification begins. In some embodiments, the second TIL amplification may be performed about 7 days after the rapid second amplification begins. In some embodiments, the second TIL amplification may be performed about 8 days after the rapid second amplification begins. In some embodiments, the second TIL amplification may be performed about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed about 10 days after the rapid second amplification begins.

In some embodiments, the rapid second amplification may be performed in a gas-permeable container using the methods of the invention (including, for example, amplification known as REP; and as indicated in FIG. 8 (particularly, for example, the process indicated in step D of FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D)). In some embodiments, TIL is expanded in the presence of IL-2, OKT-3 and feeder cells (also referred to herein as "antigen presenting cells") in a rapid second expansion. In some embodiments, TIL is expanded in the presence of IL-2, OKT-3, and feeder cells in a rapid second expansion, wherein feeder cells are added to a final concentration that is 2-fold, 2.4-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold of the concentration of feeder cells present in the beginning of the first expansion. For example, TIL can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators may include, for example, anti-CD 3 antibodies, such as about 30ng/mL OKT3, mouse monoclonal anti-CD 3 antibodies (available from Ortho-McNeil, laritan, N.J., or Meitian Biotechnology, inc. of Ornith, calif.), or UHCT-1 (available from BioLegend, san Diego, calif., U.S.A.). TIL can be amplified by including more than one antigen of the cancer (including antigenic portions thereof, e.g., epitopes) during the second amplification to induce further in vitro stimulation of TIL, which antigen can optionally be expressed by a vector, e.g., human leukocyte antigen A2 (HLa-A2) binding peptide, e.g., 0.3 μm MART-1:26-35 (27L) or gpl 00:209-217 (210M), optionally in the presence of T cell growth factors (e.g., 300IU/mL IL-2 or IL-15). Other suitable antigens may include, for example, NY-ESO-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TIL can also be rapidly amplified by restimulation with the same cancer antigen pulsed onto antigen presenting cells expressing HLA-A 2. Alternatively, the TIL may be further restimulated with, for example, example irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation is performed as part of the second amplification. In some embodiments, the second expansion is performed in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1. Mu.g/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises OKT-3 antibodies between 0.1ng/mL and 1ng/mL, between 1ng/mL and 5ng/mL, between 5ng/mL and 10ng/mL, between 10ng/mL and 20ng/mL, between 20ng/mL and 30ng/mL, between 30ng/mL and 40ng/mL, between 40ng/mL and 50ng/mL, and between 50ng/mL and 100 ng/mL. In some embodiments, the cell culture medium comprises 15ng/mL to 30ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises 30ng/mL to 60ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL OKT-3. In some embodiments, the cell culture medium comprises about 60ng/mL OKT-3. In some embodiments, the OKT-3 antibody is Moromolizumab.

In some embodiments, the medium in the rapid second amplification comprises IL-2. In some embodiments, the medium contains 6000IU/mL IL-2. In some embodiments, the medium in the rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium in the rapid second expansion comprises 7.5 x 10 ⁸ antigen presenting feeder cells per container. In some embodiments, the medium in the rapid second amplification comprises OKT-3. In some embodiments, the medium in the rapid second amplification comprises 500mL of medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS flask. In some embodiments, the medium in the rapid second amplification comprises 6000IU/mL IL-2, 60ng/mL OKT-3, and 7.5X10 ⁸ antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium and 6000IU/mL IL-2, 30 μg OKT-3, and 7.5X10 ⁸ antigen presenting feeder cells per container.

In some embodiments, the medium in the rapid second amplification comprises IL-2. In some embodiments, the medium contains 6000IU/mL IL-2. In some embodiments, the medium in the rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium comprises 5 x 10 ⁸ to 7.5 x 10 ⁸ antigen presenting feeder cells per container. In some embodiments, the medium in the rapid second amplification comprises OKT-3. In some embodiments, the medium in the rapid second amplification comprises 500mL of medium and 30 μg of OKT-3 per container. In some embodiments, the container is a G-REX-100MCS flask. In some embodiments, the medium in the rapid second expansion comprises 6000IU/mL IL-2, 60ng/mL OKT-3, and 5X 10 ⁸ to 7.5X 10 ⁸ antigen presenting feeder cells. In some embodiments, the medium in the rapid second expansion comprises 500mL of medium and 6000IU/mL IL-2, 30 μg OKT-3, and 5X 10 ⁸ to 7.5X 10 ⁸ antigen presenting feeder cells per container.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is employed as the combination during the second amplification. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included during the second amplification, including, for example, during the process according to FIG. 8 (particularly, e.g., FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D) and step D described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed as the combination during the second amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG. 8 (e.g., FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D, in particular) and step D as described herein.

In some embodiments, the second amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the second amplification medium comprises about 2IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TIL to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1: 10. about 1: 15. about 1: 20. about 1: 25. about 1: 30. about 1: 35. about 1: 40. about 1: 45. about 1: 50. about 1: 75. about 1: 100. about 1: 125. about 1: 150. about 1: 175. about 1: 200. about 1: 225. about 1: 250. about 1: 275. about 1: 300. about 1: 325. about 1: 350. about 1: 375. about 1:400 or about 1:500. in some embodiments, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1:50 and 1: 300. In some embodiments, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1:100 and 1: 200.

In some embodiments, REP and/or rapid second expansion is performed in culture flasks, wherein the bulk TIL is mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30ng/mL OKT3 anti-CD 3 antibody, and 6000IU/mL IL-2 in 150mL medium at a feeder cell concentration of at least 1.1-fold (1.1X)、1.2X、1.3X、1.4X、1.5X、1.6X、1.7X、1.8X、1.8X、2X、2.1X、2.2X、2.3X、2.4X、2.5X、2.6X、2.7X、2.8X、2.9X、3.0X、3.1X、3.2X、3.3X、3.4X、3.5X、3.6X、3.7X、3.8X、3.9X or 4.0X of the feeder cell concentration in the initial first expansion. Replacement of the medium (typically by withdrawing 2/3 of the spent medium and replacing 2/3 of the medium with an equal volume of fresh medium) until the cells are transferred to the replacement growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable containers, as discussed more fully below.

In some embodiments, the rapid second amplification (which may include a process called the REP process) is 7 to 9 days, as described in the examples and illustrations. In some embodiments, the second amplification is 7 days. In some embodiments, the second amplification is 8 days. In some embodiments, the second amplification is 9 days.

In some embodiments, the second amplification (which may include amplification called REP, and those mentioned in step D of fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)) may be performed in 500mL capacity gas-permeable flasks (G-REX-100, available from wilson wolf manufacturing company (Wilson Wolf Manufacturing Corporation) of new brayton, minnesota) with 100cm gas-permeable bottom, 5 x 10 ⁶ or 10x 10 ⁶ TILs may be cultured with PBMCs in 400mL of 50/50 medium supplemented with 5% human AB serum, 3000IU/mL IL-2, and 30ng/mL anti-CD 3 (OKT 3). G-REX-100 flasks can be incubated at 37℃in 5% CO ₂. On day 5, 250mL of supernatant may be removed, placed in a centrifuge bottle and centrifuged at 1500rpm (491 Xg) for 10 minutes. The TIL pellet may be resuspended in 150mL of fresh medium containing 5% human AB serum, 6000IU/mL IL-2 and added back to the original GREX-100 flask. When TIL is continuously amplified in GREX-100 flasks, TIL may be transferred to a larger flask, e.g., GREX-500, on day 10 or 11. Cells may be harvested on day 14 of culture. Cells may be collected on day 15 of culture. Cells may be collected on day 16 of culture. In some embodiments, the medium is replaced until the cells are transferred to the replacement growth chamber. In some embodiments, 2/3 of the medium is replaced by withdrawing spent medium and replacing it with an equal volume of fresh medium. In some embodiments, the alternative growth chamber includes GREX flasks and a gas-permeable container, as discussed more fully below.

In some embodiments, the serum-free or defined medium is CTS ^TMOpTmizer^TM T cell expansion SFM (sameir feishier technology). Any CTS ^TMOpTmizer^TM formulation may be used in the present invention. CTS ^TMOpTmizer^TM T cell expansion SFM is a combination of 1L CTS ^TMOpTmizer^TM T cell expansion basal medium with 26mL CTS ^TMOpTmizer^TM T cell expansion supplement, which is mixed together prior to use. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mM 2-mercaptoethanol.

In some embodiments, the defined medium is CTS ^TMOpTmizer^TM T cell expansion SFM (sammer feishier technology). Any CTS ^TMOpTmizer^TM formulation may be used in the present invention. CTS ^TMOpTmizer^TM T cell expansion SFM is a combination of 1L CTS ^TMOpTmizer^TM T cell expansion basal medium with 26mL CTS ^TMOpTmizer^TM T cell expansion supplement, which is mixed together prior to use. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer feier technology), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and 55mm 2-mercaptoethanol, further comprising about 1000IU/mL to about 6000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, the CTS ^TMOpTmizer^TM T cell expanded SFM is supplemented with about 3% CTS ^TM immune cell Serum Replacement (SR) (sammer technology) and about 2mM glutamine, further comprising about 6000IU/mL IL-2.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

In some embodiments, defined media (which are incorporated herein by reference) described in International patent application publication No. WO 1998/030679 and U.S. patent application publication No. US 2002/0076747A1 are suitable for use in the present invention. In this disclosure, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more components selected from the group consisting of: more than one albumin or albumin substitute, more than one amino acid, more than one vitamin, more than one transferrin or transferrin substitute, more than one antioxidant, more than one insulin or insulin substitute, more than one collagen precursor, more than one trace element, and more than one antibiotic. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β -mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: more than one amino acid, more than one vitamin, more than one transferrin or transferrin substitute, more than one antioxidant, more than one insulin or insulin substitute, more than one collagen precursor, and more than one trace element. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and compounds containing trace element moieties Ag⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺ and Zr ⁴⁺. In some embodiments, the basal cell culture medium is selected from the group consisting of: darburg's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (αmem), grange minimal essential medium (G-MEM), RPMI growth medium, and eoskov modified darburg's medium.

In some embodiments, a rapid second amplification (including an amplification called REP) is performed, further comprising a step in which TIL with excellent tumor reactivity is selected. Any selection method known in the art may be used. For example, the method described in U.S. patent application publication 2016/0010058A1, the disclosure of which is incorporated herein by reference, may be used to select TILs with excellent tumor reactivity.

Alternatively, the cell viability assay may be performed after a rapid second amplification (including an amplification known as REP amplification) using standard assays known in the art. For example, trypan blue exclusion analysis can be performed on bulk TIL samples, which selectively marks dead cells and allows survival assessment. In some embodiments, the TIL samples may be calculated and survival determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience of lorens, ma). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises 6000IU/mL IL-2, 30 μg/flask OKT-3, and 7.5X10 ⁸ antigen presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises 6000IU/mL IL-2, 30 μg/flask OKT-3, and 5X 10 ⁸ antigen presenting feeder cells (APCs) as discussed in more detail below.

In some embodiments, the rapid second amplification (e.g., according to step D) of fig. 8 (e.g., particularly fig. 8A and/or 8B and/or 8C and/or 8D) is performed in a closed system bioreactor. In some embodiments, the TIL amplification as described herein is performed using a closed system. In some embodiments, a bioreactor is employed. In some embodiments, a bioreactor is employed as the vessel. In some embodiments, the bioreactor used is, for example, G-REX-100 or G-REX-500. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-500.

In some embodiments, the step of rapidly second amplifying is split into multiple steps to achieve a longitudinal expansion of the culture scale by: (a) Performing a rapid second amplification by culturing TIL in a small scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 3 days to 7 days; and (b) effecting transfer of the TIL in the small-scale culture to a second vessel (e.g., G-REX-500-MCS vessel) that is larger than the first vessel and culturing the TIL from the small-scale culture in the larger-scale culture in the second vessel for a period of about 4 days to 7 days.

In some embodiments, the step of rapid second amplification is split into multiple steps to achieve lateral expansion of the culture scale by: (a) Performing a rapid second amplification by culturing TIL in a first small-scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 3 days to 7 days; and (b) effecting transfer and distribution of the TIL from the first small scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second vessels of equal size to the first vessel, in each of which second vessels the fraction of TIL from the first small scale culture transferred to such second vessel is cultured in the second small scale culture for a period of about 4 days to 7 days.

In some embodiments, the step of rapidly second amplifying is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Performing a rapid second amplification by culturing TIL in a small scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 3 days to 7 days; and (b) effecting transfer and distribution of TIL from the small scale culture to at least 2, 3, 4, 5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second vessels (e.g., G-REX-500MCS vessels) of larger size than the first vessel, in each of which second vessels the TIL fraction from the small scale culture transferred to such second vessel is cultured in the larger scale culture for a period of about 4 days to 7 days.

In some embodiments, the step of rapidly second amplifying is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Rapid amplification or second amplification by culturing TIL in a small scale culture in a first vessel (e.g., G-REX-100MCS vessel) for a period of about 5 days; and (b) effecting transfer and distribution of the TIL from the small-scale culture into 2, 3 or 4 second vessels (e.g., G-REX-500MCS vessels) of larger size than the first vessel, in each of which the TIL fraction from the small-scale culture transferred to such second vessel is cultured in the larger-scale culture for a period of about 6 days.

In some embodiments, each second container contains at least 10 ⁸ TILs at the time of rapid second amplification split. In some embodiments, each second container comprises at least 10 ⁸ TILs, at least 10 ⁹ TILs, or at least 10 ¹⁰ TILs at the time of the rapid or second amplification split. In one exemplary embodiment, each second container contains at least 10 ¹⁰ TILs.

In some embodiments, after completion of the rapid second amplification, the plurality of subpopulations comprises a therapeutically effective amount of TIL. In some embodiments, after rapid amplification or second amplification is completed, more than one subpopulation of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, each TIL subpopulation comprises a therapeutically effective amount of TIL after rapid amplification is completed.

In some embodiments, the rapid second amplification is performed for a period of about 3 to 7 days prior to being split into multiple steps. In some embodiments, the rapid second amplification split is performed on about day 3, day 4, day 5, day 6, or day 7 after the rapid amplification or second amplification is initiated.

In some embodiments, the rapid second amplification split is performed about day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, or day 16, day 17, or day 18 after the first amplification (i.e., pre-REP amplification) is initiated. In one exemplary embodiment, the rapid amplification or the split of the second amplification is performed about day 16 after the start of the first amplification.

In some embodiments, the rapid second amplification is further performed for a period of about 7 to 11 days after the split. In some embodiments, after the split, the rapid second amplification is further performed for a period of about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the cell culture medium for rapid second expansion prior to fractionation comprises the same components as the cell culture medium for rapid second expansion after fractionation. In some embodiments, the cell culture medium for rapid second expansion prior to fractionation comprises a different composition than the cell culture medium for rapid second expansion after fractionation.

In some embodiments, the cell culture medium for rapid second expansion prior to fractionation comprises IL-2, optionally OKT-3 and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to fractionation comprises IL-2, OKT-3 and further optional APC. In some embodiments, the cell culture medium used for rapid second expansion prior to fractionation comprises IL-2, OKT-3 and APC.

In some embodiments, the cell culture medium for rapid second expansion prior to splitting is produced by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is produced by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, OKT-3 and APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is produced by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid second expansion prior to splitting is produced by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3 and APC.

In some embodiments, the cell culture medium used for rapid second expansion after fractionation comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid second expansion after fractionation comprises IL-2 and OKT-3. In some embodiments, the cell culture medium for rapid second expansion after fractionation is produced by replacing the cell culture medium for rapid second expansion prior to fractionation with fresh medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium for rapid second expansion after fractionation is produced by replacing the cell culture medium for rapid second expansion prior to fractionation with fresh medium comprising IL-2 and OKT-3.

1. Feeder cells and antigen presenting cells

In some embodiments, the rapid second amplification procedure described herein (e.g., including amplification such as those described in step D of fig. 8 (particularly such as fig. 8A and/or 8B and/or 8C and/or 8D) and those referred to as REP) requires an excess of feeder cells during the REP TIL amplification and/or during the rapid second amplification. In many embodiments, the feeder cells are obtained from Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units of healthy blood donors. PBMC are obtained using standard methods, such as Ficoll-Paque gradient separation.

In some embodiments, PBMCs are considered replication incompetent and acceptable for the TIL expansion procedure described herein if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of second expansion (i.e., the starting day of second expansion).

In some embodiments, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedure described herein if the total number of living cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 is not increased compared to the initial number of living cells placed in culture on day 0 of REP and/or day 0 of second expansion (i.e., the starting day of second expansion). In some embodiments, PBMC are cultured in the presence of 30ng/mL OKT3 antibody and 3000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 60ng/mL OKT3 antibody and 6000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 60ng/mL OKT3 antibody and 3000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 30ng/mL OKT3 antibody and 6000IU/mL IL-2.

In some embodiments, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedure described herein if the total number of living cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 is not increased compared to the initial number of living cells placed in culture on day 0 of REP and/or day 0 of second expansion (i.e., the starting day of second expansion). In some embodiments, PBMC are cultured in the presence of 30 to 60ng/mL OKT3 antibody and 1000 to 6000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 30 to 60ng/mL OKT3 antibody and 2000 to 5000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 30 to 60ng/mL OKT3 antibody and 2000 to 4000IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 30 to 60ng/mL OKT3 antibody and 2500 to 3500IU/mL IL-2. In some embodiments, PBMC are cultured in the presence of 30 to 60ng/mL OKT3 antibody and 6000IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second amplification is about 1: 10. about 1: 25. about 1: 50. about 1: 100. about 1: 125. about 1: 150. about 1: 175. about 1: 200. about 1: 225. about 1: 250. about 1: 275. about 1: 300. about 1: 325. about 1: 350. about 1: 375. about 1:400 or about 1:500. in some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1: 300. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1: 200.

In some embodiments, the second expansion procedure described herein requires a ratio of about 5×10 ⁸ feeder cells to about 100×10 ⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of about 7.5×10 ⁸ feeder cells to about 100×10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of about 5×10 ⁸ feeder cells to about 50×10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of about 7.5X10 ⁸ feeder cells to about 50X 10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires about 5×10 ⁸ feeder cells with about 25×10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires about 7.5X10 ⁸ feeder cells with about 25X 10 ⁶ TILs. In other embodiments, the rapid second expansion requires a 2-fold number of feeder cells for the rapid second expansion. In other embodiments, when about 2.5X10 ⁸ feeder cells are required to initiate the first expansion described herein, about 5X 10 ⁸ feeder cells are required for the rapid second expansion. In other embodiments, when about 2.5X10 ⁸ feeder cells are required to initiate the first expansion described herein, about 7.5X10 ⁸ feeder cells are required to rapidly expand the second expansion. In other embodiments, rapid second expansion requires 2-fold (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X number of feeder cells to initiate the first expansion.

In some embodiments, the rapid second expansion procedure described herein requires an excess of feeder cells during the rapid second expansion. In many embodiments, the feeder cells are obtained from Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units of allogeneic healthy blood donors. PBMC are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aapcs) are used in place of PBMCs. In some embodiments, PBMCs are added to the rapid second amplification at 2 times the concentration of PBMCs added to the initial first amplification.

In some embodiments, artificial antigen presenting cells are used in the rapid second expansion in place of or in combination with PBMCs.

2. Cytokines and other additives

The rapid second amplification methods described herein generally use media with high doses of cytokines (particularly IL-2), as known in the art.

Or a combination of cytokines to rapidly second amplify TIL is also possible, as described in U.S. patent application publication No. US2017/0107490A1, using a combination of two or more IL-2, IL-15 and IL-21, the disclosures of which are incorporated herein by reference. Thus, it is possible to combine IL-2 with IL-15, IL-2 with IL-21, IL-15 with IL-21, and IL-2, IL-15 with IL-21, the latter having particular utility in many embodiments. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, and in particular T cells as described therein.

In some embodiments, step D (particularly from, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) may also comprise adding OKT-3 antibody or moromiab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include adding a 4-1BB agonist to the medium, as described elsewhere herein. In some embodiments, step D (particularly from, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) may also comprise adding an OX-40 agonist to the culture medium, as described elsewhere herein. Furthermore, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR) -gamma agonists, e.g. thiazolidinedione compounds, may be used in the medium during step D (particularly from e.g. fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D), as described in US patent application publication No. US2019/0307796 A1, the disclosure of which is incorporated herein by reference.

E. step E: collecting TIL

After the rapid second expansion step, the cells may be collected. In some embodiments, TIL is collected after one, two, three, four or more amplification steps such as provided in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, TIL is collected after two amplification steps provided, for example, in fig. 8 (in particular, for example, fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL is collected after two amplification steps (one initial first amplification and one rapid second amplification) such as provided in fig. 8 (in particular, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D).

The TIL may be collected in any suitable and sterile manner, including, for example, centrifugation. Methods of collecting TIL are well known in the art and any such known methods may be used with the process of the present invention. In some embodiments, the TIL is collected using an automated system.

In some embodiments, step E according to fig. 8 (e.g., fig. 8A and/or 8B and/or 8C and/or 8D in particular) is performed according to the processes described herein. In some embodiments, the containment system is accessed through a syringe under sterile conditions to maintain sterility and containment properties of the system. In some embodiments, a closed system as described herein is employed.

In some embodiments, TILs are collected according to the methods described herein. In some embodiments, TIL between days 14 and 16 is collected using the methods as described herein. In some embodiments, TIL is collected on day 14 using the methods as described herein. In some embodiments, TIL is collected on day 15 using the methods as described herein. In some embodiments, TIL is collected on day 16 using the methods as described herein.

F. Step F: final formulation and transfer to infusion container

After steps a to E are completed as provided in an exemplary sequence in fig. 8 (e.g., fig. 8A and/or 8B and/or 8C and/or 8D in particular) and as described above and herein, the cells are transferred into a container for administration to a patient, e.g., an infusion bag or sterile vial. In some embodiments, once a sufficient number of TILs are obtained for treatment using the amplification methods described above, they are transferred to a container for administration to a patient.

In some embodiments, TIL amplified using the methods of the present disclosure is administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. Amplified TIL as disclosed herein may be administered by any suitable route as known in the art. In some embodiments, the TIL is administered as a single intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

VIII, other Gen 2, gen 3 and other examples of TIL manufacturing Process

Pbmc feeder cell ratio

In some embodiments, the medium used in the amplification methods described herein (see, e.g., fig. 8 (e.g., particularly fig. 8A and/or 8B and/or 8C and/or 8D)) comprises an anti-CD 3 antibody, e.g., OKT-3. The combination of anti-CD 3 antibodies with IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length antibodies, which are generally preferred, as well as Fab and F (ab') 2 fragments; see, e.g., tsoukas et al, journal of immunology 1985,135,1719, which is hereby incorporated by reference in its entirety.

In some embodiments, the number of PBMC feeder cell layers is calculated as follows:

A.T cell volume (diameter 10 μm): v= (4/3) pi r ³＝523.6μm³

B. G-REX-100 (M) volume with a height of 40 μm (4 cells): v= (4/3) pi r ³＝4×10¹²μm³

C. Cell number required to fill volume B: 4X 10 ¹²μm³/523.6μm³＝7.6×10⁸μm³*0.64＝4.86×10⁸

D. Cell number that can be optimally activated in 4D space: 4.86×10 ⁸/24＝20.25×10⁶

E. Feeder cells and TIL numbers extrapolated to G-REX-500: TIL:100×10 ⁶ and feeder cells: 2.5X10 ⁹

In this calculation, the approximate number of monocytes required to provide TIL-activated icosahedral geometry in a cylinder with a 100cm ² base was used. The experimental result of the calculated activation of the threshold T-cells was about 5X 10 ⁸, which is closely related to NCI experimental data, as described in Jin et al, J.Immunotherapy, 2012,35,283-292. In (C), the multiplier (0.64) is the random packing density of the equivalent spheres, calculated by Jaeger and Nagel, science 1992,255,1523-3. In (D), the divisor 24 is the number or "newton number" of equivalent spheres in 4-dimensional space that can contact similar objects, as described in Musin, russian mathematical comment (russ.math.surv.), 2003,58,794-795.

In some embodiments, the number of exogenously supplied antigen presenting feeder cells during initiation of the first expansion is about half the number of exogenously supplied antigen presenting feeder cells during the rapid second expansion. In certain embodiments, the method comprises initiating the first expansion in a cell culture medium comprising about 50% fewer antigen presenting cells than the cell culture medium of the rapid second expansion.

In other embodiments, the number of exogenously supplied antigen presenting feeder cells (APCs) during the rapid second amplification is greater than the number of exogenously supplied APCs during the initiation of the first amplification.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 20:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 10:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 9:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 8:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 7:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 6:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 5:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 4:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 3:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.9:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.8:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.7:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.6:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.5:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.4:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.3:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.2:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2.1:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 1.1:1 to just or about 2:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 10:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 5:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 4:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 3:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.9:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.8:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.7:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.6:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.5:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.4:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.3:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.2:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is selected from the range of just or about 2:1 to just or about 2.1:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is exactly or about 2:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initiation of the first amplification is just or about 1.1:1、1.2:1、1.3:1、1.4:1、1.5:1、1.6:1、1.7:1、1.8:1、1.9:1、2:1、2.1:1、2.2:1、2.3:1、2.4:1、2.5:1、2.6:1、2.7:1、2.8:1、2.9:1、3:1、3.1:1、3.2:1、3.3:1、3.4:1、3.5:1、3.6:1、3.7:1、3.8:1、3.9:1、4:1、4.1:1、4.2:1、4.3:1、4.4:1、4.5:1、4.6:1、4.7:1、4.8:1、4.9:1 or 5:1.

In other embodiments, the number of APCs exogenously supplied during initiation of the first amplification is exactly or about 1×10⁸、1.1×10⁸、1.2×10⁸、1.3×10⁸、1.4×10⁸、1.5×10⁸、1.6×10⁸、1.7×10⁸、1.8×10⁸、1.9×10⁸、2×10⁸、2.1×10⁸、2.2×10⁸、2.3×10⁸、2.4×10⁸、2.5×10⁸、2.6×10⁸、2.7×10⁸、2.8×10⁸、2.9×10⁸、3×10⁸、3.1×10⁸、3.2×10⁸、3.3×10⁸、3.4×10⁸ or 3.5x10 ⁸ APCs, and the number of APCs exogenously supplied during rapid second amplification is exactly or about 3.5×10⁸、3.6×10⁸、3.7×10⁸、3.8×10⁸、3.9×10⁸、4×10⁸、4.1×10⁸、4.2×10⁸、4.3×10⁸、4.4×10⁸、4.5×10⁸、4.6×10⁸、4.7×10⁸、4.8×10⁸、4.9×10⁸、5×10⁸、5.1×10⁸、5.2×10⁸、5.3×10⁸、5.4×10⁸、5.5×10⁸、5.6×10⁸、5.7×10⁸、5.8×10⁸、5.9×10⁸、6×10⁸、6.1×10⁸、6.2×10⁸、6.3×10⁸、6.4×10⁸、6.5×10⁸、6.6×10⁸、6.7×10⁸、6.8×10⁸、6.9×10⁸、7×10⁸、7.1×10⁸、7.2×10⁸、7.3×10⁸、7.4×10⁸、7.5×10⁸、7.6×10⁸、7.7×10⁸、7.8×10⁸、7.9×10⁸、8×10⁸、8.1×10⁸、8.2×10⁸、8.3×10⁸、8.4×10⁸、8.5×10⁸、8.6×10⁸、8.7×10⁸、8.8×10⁸、8.9×10⁸、9×10⁸、9.1×10⁸、9.2×10⁸、9.3×10⁸、9.4×10⁸、9.5×10⁸、9.6×10⁸、9.7×10⁸、9.8×10⁸、9.9×10⁸ or 1 x 10 ⁹ APCs.

In other embodiments, the number of APCs exogenously supplied during initiation of the first amplification is selected from the range of exactly or about 1.5x10 ⁸ APCs to exactly or about 3 x 10 ⁸ APCs, and the number of APCs exogenously supplied during rapid second amplification is selected from the range of exactly or about 4 x 10 ⁸ APCs to exactly or about 7.5x10 ⁸ APCs.

In other embodiments, the number of APCs exogenously supplied during initiation of the first amplification is selected from the range of just or about 2×10 ⁸ APCs to just or about 2.5×10 ⁸ APCs, and the number of APCs exogenously supplied during rapid second amplification is selected from the range of just or about 4.5×10 ⁸ APCs to just or about 5.5×10 ⁸ APCs.

In other embodiments, the number of APCs exogenously supplied during initiation of the first amplification is just or about 2.5x10 ⁸ APCs, and the number of APCs exogenously supplied during the rapid second amplification is just or about 5 x 10 ⁸ APCs.

In some embodiments, the number of APCs (including, e.g., PBMCs) added on day 0 of initiation of the first amplification is about half the number of PBMCs added on day 7 of initiation of the first amplification (e.g., day 7 of the method). In certain embodiments, the method comprises adding antigen presenting cells to the first TIL population on day 0 of initiation of the first expansion and adding antigen presenting cells to the second TIL population on day 7, wherein the number of antigen presenting cells added on day 0 is about 50% of the number of antigen presenting cells added on day 7 of initiation of the first expansion (e.g., day 7 of the method).

In other embodiments, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification is greater than the number of PBMCs exogenously supplied on day 0 of initiation of first amplification.

In other embodiments, the APCs exogenously supplied at the start of the first amplification are inoculated in culture flasks at a density selected from the range of just or about 1.0X10 ⁶ APCs/cm ² to just or about 4.5X10 ⁶ APCs/cm ².

In other embodiments, the APCs exogenously supplied at the start of the first amplification are inoculated in culture flasks at a density selected from the range of just or about 1.5X10 ⁶ APCs/cm ² to just or about 3.5X10 ⁶ APCs/cm ².

In other embodiments, the APCs exogenously supplied at the start of the first amplification are inoculated in culture flasks at a density selected from the range of just or about 2 x 10 ⁶ APCs/cm ² to just or about 3 x 10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied exogenously at the start of the first amplification are inoculated in culture flasks at a density of just or about 2 x 10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied exogenously at the start of the first amplification are inoculated in culture flasks at a density of just or about 1.0×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶ or 4.5x10 ⁶ APCs/cm ².

In other embodiments, APCs supplied at the rapid second amplification source are inoculated in culture flasks at a density selected from the range of just or about 2.5X10 ⁶ APCs/cm ² to just or about 7.5X10 ⁶ APCs/cm ².

In other embodiments, APCs supplied at the rapid second amplification source are inoculated in culture flasks at a density selected from the range of just or about 3.5X10 ⁶ APCs/cm ² to just or about 6.0X10 ⁶ APCs/cm ².

In other embodiments, APCs supplied at the rapid second amplification source are inoculated in culture flasks at a density selected from the range of just or about 4.0X10 ⁶ APCs/cm ² to just or about 5.5X10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied at the rapid second amplification source are inoculated in the flask at a density selected from the range of just or about 4.0X10 ⁶ APCs/cm ².

In other embodiments, APCs supplied at the rapid second amplification source are inoculated in culture flasks at a density of just or about 2.5X10 ⁶ APCs/cm ²、2.6×10⁶ APCs/cm ²、2.7×10⁶ APC/cm²、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.1×10⁶、6.2×10⁶、6.3×10⁶、6.4×10⁶、6.5×10⁶、6.6×10⁶、6.7×10⁶、6.8×10⁶、6.9×10⁶、7×10⁶、7.1×10⁶、7.2×10⁶、7.3×10⁶、7.4×10⁶ or 7.5X10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied at the initial first amplification source are inoculated in the flask at a density of just or about 1.0×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶ or 4.5X10 ⁶ APCs/cm ², and the APCs supplied at the rapid second amplification source are inoculated in the flask at a density of just or about 2.5X10 ⁶ APCs/cm ²、2.6×10⁶ APCs/cm ²、2.7×10⁶ APC/cm²、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.1×10⁶、6.2×10⁶、6.3×10⁶、6.4×10⁶、6.5×10⁶、6.6×10⁶、6.7×10⁶、6.8×10⁶、6.9×10⁶、7×10⁶、7.1×10⁶、7.2×10⁶、7.3×10⁶、7.4×10⁶ or 7.5X10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied at the initiation of the first amplification exogenous are inoculated in the culture flask at a density selected from the range of just or about 1.0X10 ⁶ APCs/cm ² to just or about 4.5X10 ⁶ APCs/cm ² and the APCs supplied at the rapid second amplification exogenous are inoculated in the culture flask at a density selected from the range of just or about 2.5X10 ⁶ APCs/cm ² to just or about 7.5X10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied at the initiation of the first amplification exogenous are inoculated in the culture flask at a density selected from the range of just or about 1.5X10 ⁶ APCs/cm ² to just or about 3.5X10 ⁶ APCs/cm ² and the APCs supplied at the rapid second amplification exogenous are inoculated in the culture flask at a density selected from the range of just or about 3.5X10 ⁶ APCs/cm ² to just or about 6X 10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied at the initiation of the first amplification exogenous are inoculated in the culture flask at a density selected from the range of just or about 2X 10 ⁶ APCs/cm ² to just or about 3X 10 ⁶ APCs/cm ², and the APCs supplied at the rapid second amplification exogenous are inoculated in the culture flask at a density selected from the range of just or about 4X 10 ⁶ APCs/cm ² to just or about 5.5X 10 ⁶ APCs/cm ².

In other embodiments, the APCs supplied at the initial first amplification source are inoculated in the flask at a density of just or about 2X 10 ⁶ APCs/cm ² and the APCs supplied at the rapid second amplification source are inoculated in the flask at a density of just or about 4X 10 ⁶ APCs/cm ².

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of PBMCs exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 20:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of PBMCs exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 10:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of PBMCs exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 9:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 8:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 7:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 6:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 5:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 4:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 3:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.9:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.8:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.7:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.6:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.5:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.4:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.3:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.2:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2.1:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 1.1:1 to just or about 2:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 10:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 5:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 4:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 3:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.9:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.8:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.7:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.6:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.5:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.4:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.3:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.2:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is selected from the range of just or about 2:1 to just or about 2.1:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is just or about 2:1.

In other embodiments, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification is just or about 1.1:1、1.2:1、1.3:1、1.4:1、1.5:1、1.6:1、1.7:1、1.8:1、1.9:1、2:1、2.1:1、2.2:1、2.3:1、2.4:1、2.5:1、2.6:1、2.7:1、2.8:1、2.9:1、3:1、3.1:1、3.2:1、3.3:1、3.4:1、3.5:1、3.6:1、3.7:1、3.8:1、3.9:1、4:1、4.1:1、4.2:1、4.3:1、4.4:1、4.5:1、4.6:1、4.7:1、4.8:1、4.9:1 or 5:1.

In other embodiments, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of the first amplification is exactly or about 1×10⁸、1.1×10⁸、1.2×10⁸、1.3×10⁸、1.4×10⁸、1.5×10⁸、1.6×10⁸、1.7×10⁸、1.8×10⁸、1.9×10⁸、2×10⁸、2.1×10⁸、2.2×10⁸、2.3×10⁸、2.4×10⁸、2.5×10⁸、2.6×10⁸、2.7×10⁸、2.8×10⁸、2.9×10⁸、3×10⁸、3.1×10⁸、3.2×10⁸、3.3×10⁸、3.4×10⁸ or 3.5x10 ⁸ APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is exactly or about 3.5×10⁸、3.6×10⁸、3.7×10⁸、3.8×10⁸、3.9×10⁸、4×10⁸、4.1×10⁸、4.2×10⁸、4.3×10⁸、4.4×10⁸、4.5×10⁸、4.6×10⁸、4.7×10⁸、4.8×10⁸、4.9×10⁸、5×10⁸、5.1×10⁸、5.2×10⁸、5.3×10⁸、5.4×10⁸、5.5×10⁸、5.6×10⁸、5.7×10⁸、5.8×10⁸、5.9×10⁸、6×10⁸、6.1×10⁸、6.2×10⁸、6.3×10⁸、6.4×10⁸、6.5×10⁸、6.6×10⁸、6.7×10⁸、6.8×10⁸、6.9×10⁸、7×10⁸、7.1×10⁸、7.2×10⁸、7.3×10⁸、7.4×10⁸、7.5×10⁸、7.6×10⁸、7.7×10⁸、7.8×10⁸、7.9×10⁸、8×10⁸、8.1×10⁸、8.2×10⁸、8.3×10⁸、8.4×10⁸、8.5×10⁸、8.6×10⁸、8.7×10⁸、8.8×10⁸、8.9×10⁸、9×10⁸、9.1×10⁸、9.2×10⁸、9.3×10⁸、9.4×10⁸、9.5×10⁸、9.6×10⁸、9.7×10⁸、9.8×10⁸、9.9×10⁸ or 1 x 10 ⁹ APCs (including, e.g., PBMCs).

In other embodiments, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of the first amplification is selected from the range of exactly or about 1×10 ⁸ APCs (including, e.g., PBMCs) to exactly or about 3.5×10 ⁸ APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification is selected from the range of exactly or about 3.5×10 ⁸ APCs (including, e.g., PBMCs) to exactly or about 1×10 ⁹ APCs (including, e.g., PBMCs).

In other embodiments, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of the first amplification is selected from the range of just or about 1.5×10 ⁸ APCs (including, e.g., PBMCs) to just or about 3×10 ⁸ APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is selected from the range of just or about 4×10 ⁸ APCs (including, e.g., PBMCs) to just or about 7.5×10 ⁸ APCs (including, e.g., PBMCs).

In other embodiments, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of the first amplification is selected from the range of exactly or about 2 x 10 ⁸ APCs (including, e.g., PBMCs) to exactly or about 2.5 x 10 ⁸ APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification is selected from the range of exactly or about 4.5 x 10 ⁸ APCs (including, e.g., PBMCs) to exactly or about 5.5 x 10 ⁸ APCs (including, e.g., PBMCs).

In other embodiments, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of the first amplification is exactly or about 2.5×10 ⁸ APCs (including, e.g., PBMCs) and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification is exactly or about 5×10 ⁸ APCs (including, e.g., PBMCs).

In some embodiments, the number of APC (including, e.g., PBMCs) added on day 0 of initiation of the first amplification is about half the number of APC (including, e.g., PBMCs) added on day 7 of the rapid second amplification. In certain embodiments, the method comprises adding an antigen presenting cell layer to the first TIL population on day 0 of initiating the first expansion and adding an antigen presenting cell layer to the second TIL population on day 7, wherein the number of antigen presenting cell layers added on day 0 is about 50% of the number of antigen presenting cell layers added on day 7.

In other embodiments, the number of layers of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of rapid second amplification is greater than the number of layers of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiation of first amplification.

In other embodiments, day 0 of the initial first expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 2 cell layers, and day 7 of the rapid second expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 4 cell layers.

In other embodiments, day 0 of the initial first expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 1 cell layer, and day 7 of the rapid second expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 3 cell layers.

In other embodiments, day 0 of the initial first expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers, and day 7 of the rapid second expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 3 cell layers.

In other embodiments, day 0 of the initial first expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 1 cell layer, and day 7 of the rapid second expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 2 cell layers.

In other embodiments, day 0 of the initial first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers, and day 7 of the rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9 or 8 cell layers.

In other embodiments, day 0 of the initial first expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from just or about 1 cell layer to just or about 2 cell layers, and day 7 of the rapid second expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, day 0 of the initial first expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from just or about 2 cell layers to just or about 3 cell layers, and day 7 of the rapid second expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of the initial first expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 2 cell layers, and day 7 of the rapid second expansion is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of the initial first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 1, 2, or 3 cell layers, and day 7 of the rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of just or about 3, 4,5, 6, 7, 8, 9, or 10 cell layers.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is selected from the range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness that is equal to the number of layers of the first APC (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness that is equal to the number of layers of the second APC (including, e.g., PBMCs), wherein the ratio of the number of layers of the first APC (including, e.g., PBMCs) to the number of layers of the second APC (including, e.g., PBMCs) is exactly or about 1:2.

In other embodiments, day 0 of initiating the first amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a first average thickness equal to the number of layers of first APCs (including, e.g., PBMCs), and day 7 of rapid second amplification is performed in the presence of layered APCs (including, e.g., PBMCs) having a second average thickness equal to the number of layers of second APCs (including, e.g., PBMCs), wherein the ratio of the number of layers of first APCs (including, e.g., PBMCs) to the number of layers of second APCs (including, e.g., PBMCs) is selected from just or about 1:1.1、1:1.2、1:1.3、1:1.4、1:1.5、1:1.6、1:1.7、1:1.8、1:1.9、1:2、1:2.1、1:2.2、1:2.3、1:2.4、1:2.5、1:2.6、1:2.7、1:2.8、1:2.9、1:3、1:3.1、1:3.2、1:3.3、1:3.4、1:3.5、1:3.6、1:3.7、1:3.8、1:3.9、1:4、1:4.1、1:4.2、1:4.3、1:4.4、1:4.5、1:4.6、1:4.7、1:4.8、1:4.9、1:5、1:5.1、1:5.2、1:5.3、1:5.4、1:5.5、1:5.6、1:5.7、1:5.8、1:5.9、1:6、1:6.1、1:6.2、1:6.3、1:6.4、1:6.5、1:6.6、1:6.7、1:6.8、1:6.9、1:7、1:7.1、1:7.2、1:7.3、1:7.4、1:7.5、1:7.6、1:7.7、1:7.8、1:7.9、1:8、1:8.1、1:8.2、1:8.3、1:8.4、1:8.5、1:8.6、1:8.7、1:8.8、1:8.9、1:9、1:9.1、1:9.2、1:9.3、1:9.4、1:9.5、1:9.6、1:9.7、1:9.8、1:9.9 or 1:10.

In some embodiments, the number of APCs in the initial first amplification is selected from the range of about 1.0x10 ⁶ APCs/cm ² to about 4.5x10 ⁶ APCs/cm ² and the number of APCs in the rapid second amplification is selected from the range of about 2.5x10 ⁶ APCs/cm ² to about 7.5x10 ⁶ APCs/cm ².

In some embodiments, the number of APCs in the initial first amplification is selected from the range of about 1.5x10 ⁶ APCs/cm ² to about 3.5x10 ⁶ APCs/cm ² and the number of APCs in the rapid second amplification is selected from the range of about 3.5x10 ⁶ APCs/cm ² to about 6.0x10 ⁶ APCs/cm ².

In some embodiments, the number of APCs in the initial first amplification is selected from the range of about 2.0x10 ⁶ APCs/cm ² to about 3.0x10 ⁶ APCs/cm ² and the number of APCs in the rapid second amplification is selected from the range of about 4.0x10 ⁶ APCs/cm ² to about 5.5x10 ⁶ APCs/cm ².

A. Optional cell culture Medium Components

1. Anti-CD 3 antibodies

In some embodiments, the medium used in the amplification methods described herein (see, e.g., fig. 1 and 8 (e.g., fig. 8B in particular)) comprises an anti-CD 3 antibody. The combination of anti-CD 3 antibodies with IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length antibodies, which are generally preferred, as well as Fab and F (ab') 2 fragments; see, e.g., tsoukas et al, journal of immunology 1985,135,1719, which is hereby incorporated by reference in its entirety.

As will be appreciated by those skilled in the art, some suitable anti-human CD3 antibodies may be used in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD 3 antibody, moromolizumab (available from Ortho-McNeil, inc. of Laritan, N.J., or Meter-Tian-Mi Biotechnology, inc. of Orben, calif.) is used. See table 1.

As will be appreciated by those skilled in the art, some suitable anti-human CD3 antibodies may be used in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD 3 antibody, moromolizumab (available from Ortho-McNeil, inc. of Laritan, N.J., or Meter-Tian-Mi Biotechnology, inc. of Orben, calif.) is used.

2.4-1BB (CD 137) agonists

In some embodiments, the cell culture medium that initiates the first expansion and/or the rapid second expansion comprises a TNFRSF agonist. In some embodiments, the TNFRSF agonist is a 4-1BB (CD 137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1 BB. The 4-1BB agonist or 4-1BB binding molecule may comprise an immunoglobulin heavy chain of any isotype (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass of immunoglobulin molecule. The 4-1BB agonist or 4-1BB binding molecule may have a heavy chain and a light chain. As used herein, the term binding molecule also includes antibodies (including full length antibodies); monoclonal antibodies (including full length monoclonal antibodies); a polyclonal antibody; multispecific antibodies (e.g., bispecific antibodies); a human, humanized or chimeric antibody; and antibody fragments, such as Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, epitope-binding fragments of any of the foregoing, and engineered forms of antibodies that bind to 4-1BB, such as scFv molecules. In some embodiments, the 4-1BB agonist is an antigen binding protein of a fully human antibody. In some embodiments, the 4-1BB agonist is an antigen binding protein of a humanized antibody. In some embodiments, 4-1BB agonists useful in the methods and compositions disclosed herein include anti-4-1 BB antibodies, human anti-4-1 BB antibodies, mouse anti-4-1 BB antibodies, mammalian anti-4-1 BB antibodies, monoclonal anti-4-1 BB antibodies, polyclonal anti-4-1 BB antibodies, chimeric anti-4-1 BB antibodies, anti-4-1 BB adnectin (adnectin), anti-4-1 BB domain antibodies, single chain anti-4-1 BB fragments, heavy chain anti-4-1 BB fragments, light chain anti-4-1 BB fragments, anti-4-1 BB fusion proteins, and fragments, derivatives, conjugates, variants, or biological analogs thereof. The agonistic anti-4-1 BB antibody is known to induce a strong immune response. Lee et al, public science library, complex (PLOS One) 2013,8, e69677. In some embodiments, the 4-1BB agonist is an agonistic anti-4-1 BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In some embodiments, the 4-1BB agonist is EU-101 (Eutilex co.ltd.), nivolumab or nivolumab, or a fragment, derivative, conjugate, variant, or biological analog thereof. In some embodiments, the 4-1BB agonist is wutuzumab or wuruituzumab or a fragment, derivative, conjugate, variant, or biological analog thereof.

In some embodiments, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In some embodiments, a multimeric 4-1BB agonist, such as a trimeric or hexameric 4-1BB agonist (having three or six ligand binding domains), may induce superior receptor (4-1 BBL) clustering and internal cell signaling complex formation compared to an agonistic monoclonal antibody that typically has two ligand binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins are described, for example, in Gieffers et al, molecular cancer Therapeutics (mol. Cancer) 2013,12,2735-47.

The agonistic 4-1BB antibody and fusion proteins are known to induce a strong immune response. In some embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent cell phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates Complement Dependent Cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates the functionality of the Fc region.

In some embodiments, the 4-1BB agonist is characterized as binding to human 4-1BB (SEQ ID NO: 40) with high affinity and agonistic activity. In some embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 40). In some embodiments, the 4-1BB agonist is a binding molecule that binds to murine 4-1BB (SEQ ID NO: 41). The amino acid sequences of the 4-1BB antigens to which the 4-1BB agonists or binding molecules bind are summarized in Table 13.

Table 13: amino acid sequence of 4-1BB antigen.

In some embodiments, the described compositions, processes, and methods include 4-1BB agonists that bind to human or murine 4-1BB at a K _D of about 100pM or less, to human or murine 4-1BB at a K _D of about 90pM or less, to human or murine 4-1BB at a K _D of about 80pM or less, to human or murine 4-1BB at a K _D of about 70pM or less, to human or murine 4-1BB at a K _D of about 60pM or less, to human or murine 4-1BB at a K _D of about 50pM or less, to human or murine 4-1BB at a K _D of about 40pM or less, or to human or murine 4-1BB at a K _D of about 30pM or less.

In some embodiments, the described compositions, processes, and methods include a 4-1BB agonist that binds to human or murine 4-1BB at a k _assoc of about 7.5X10 ⁵/M.s or faster, binds to human or murine 4-1BB at a k _assoc of about 7.5X10 ⁵ 1/M.s or faster, binds to human or murine 4-1BB at a k _assoc of about 8X 10 ⁵ l/M.s or faster, binds to human or murine 4-1BB at a k _assoc of about 8.5X10 ⁵ 1/M.s or faster, binds to human or murine 4-1BB at a k _assoc of about 9X 10 ⁵ 1/M.s or faster, binds to human or murine 4-1BB at a k _assoc of about 9.5X10 ⁵/M.s or faster, or binds to human or murine 4-1BB at a k _assoc of about 8.5X10.1/M.s or faster.

In some embodiments, the compositions, processes, and methods described include a 4-1BB agonist, the 4-1BB agonist binds to human or murine 4-1BB at about 2X 10 ^-5/s or slower k _dissoc, to human or murine 4-1BB at about 2.1X10 ^-5/s or slower k _dissoc, to human or murine 4-1BB at about 2.2X10 ^-5 1/s or slower k _dissoc, to human or murine 4-1BB at about 2.3X10 ^-5/s or slower k _dissoc, to human or murine 4-1BB at about 2.4X10 ^-5/s or slower k _dissoc, to human or murine 4-1BB at about 2.5X10 ^-5/s or slower k _dissoc, to human or murine 4-1BB at about 2.6X10 ^-5 1/s or slower k _dissoc, or to human or murine 4-1BB at about 2.7X10 ^-5/s or slower k _dissoc, to human or murine 4-1/s or about 2.4X10/s or slower k _dissoc/s or about 2.5X10/s or slower k _dissoc, to human or murine 4-1BB at about 2.5X10.6X10/s or slower k 5235, to human or murine 4-1 BB.

In some embodiments, the described compositions, processes, and methods include a 4-1BB agonist that binds to human or murine 4-1BB with an IC ₅₀ of about 10nM or less, to human or murine 4-1BB with an IC ₅₀ of about 9nM or less, to human or murine 4-1BB with an IC ₅₀ of about 8nM or less, to human or murine 4-1BB with an IC ₅₀ of about 7nM or less, to human or murine 4-1BB with an IC ₅₀ of about 6nM or less, to human or murine 4-1BB with an IC ₅₀ of about 5nM or less, to human or murine 4-1BB with an IC ₅₀ of about 4nM or less, to human or murine 4-1BB with an IC ₅₀ of about 3nM or less, to human or murine 4-1BB with an IC ₅₀ of about 2nM or less, or to human or murine 4-1BB with an IC ₅₀ of about 1nM or less.

In some embodiments, the 4-1BB agonist is WUGUMAXMAZU (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant, or biological analog thereof. Wu Tumu mab is available from the company Pfizer, inc. Wu Tumu monoclonal antibody is immunoglobulin G2-lambda anti [ Chile TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9,4-1BB, T cell antigen ILA, CD 137) ] Chile (fully human) monoclonal antibody. The amino acid sequence of Wu Tumu mab is set forth in table 14. Wu Tumu the mab comprises glycosylation sites at Asn59 and Asn 292; heavy chain intra-chain disulfide bonds at positions 22-96 (V _H-V_L)、143-199(C_H1-CL)、256-316(C_H 2) and 362-420 (C _H); light chain intra-chain disulfide bonds at positions 22'-87' (V _H-V_L) and 136'-195' (C _H 1-CL); inter-chain heavy chain-heavy chain disulfide bonds at IgG2A isomer positions 218-218, 219-219, 222-222, and 225-225, at IgG2A/B isomer positions 218-130, 219-219, 222-222, and 225-225, and at IgG2B isomer positions 219-130 (2), 222-222, and 225-225; and inter-chain heavy chain-light chain disulfide bonds at IgG2A isoform positions 130-213 '(2), igG2A/B isoform positions 218-213' and 130-213', and at IgG2B isoform positions 218-213' (2). The preparation and properties of Wu Tumu mab and variants and fragments thereof are described in U.S. Pat. nos. 8,821,867, 8,337,850 and 9,468,678 and international patent application publication No. WO 2012/0325433 A1, the respective disclosures of which are incorporated herein by reference. The preclinical characterization of Wu Tumu mab is described in Fisher et al, cancer immunology and immunotherapy (Cancer Immunolog. & immunother.) 2012,61,1721-33. Current clinical trials of ulipristal in a variety of blood and solid tumor indications include the national institutes of health, clinicaltrias, gov, identification number NCT02444793, NCT01307267, NCT02315066 and NCT02554812.

In some embodiments, the 4-1BB agonist comprises the sequence of SEQ ID NO:42 and the heavy chain shown in SEQ ID NO: 43. In some embodiments, the 4-1BB agonist comprises a sequence having the amino acid sequence of SEQ ID NO:42 and SEQ ID NO:43, or an antigen binding fragment, fab fragment, single chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 99% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 98% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 97% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 96% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 95% identity.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of Wu Tumu mab. In some embodiments, the 4-1BB agonist heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:44, and the 4-1BB agonist light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:45, and conservative amino acid substitutions thereof. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:44 and SEQ ID NO:45 has at least 99% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:44 and SEQ ID NO:45 has at least 98% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:44 and SEQ ID NO:45 has at least 97% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:44 and SEQ ID NO:45 has at least 96% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:44 and SEQ ID NO:45 has at least 95% identity. In some embodiments, the 4-1BB agonist comprises: scFv antibodies comprising V _H and V _L regions, each of which hybridizes to SEQ ID NO:44 and SEQ ID NO:45 has at least 99% identity.

In some embodiments, the 4-1BB agonist comprises a sequence having the amino acid sequence of SEQ ID NO: 46. SEQ ID NO:47 and SEQ ID NO:48 and the heavy chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 49. SEQ ID NO:50 and SEQ ID NO:51 and the light chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by a drug administration with reference to Ustilbenmumab. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody that comprises an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference drug or reference biologic that comprises one or more post-translational modifications as compared to the reference drug or reference biologic that is wushuzumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a different formulation than the formulation of the reference drug or the reference biological product, wherein the reference drug or the reference biological product is Ubbelohde mab. The 4-1BB agonist antibodies may be obtained from a drug administration, such as U.S. FDA and/or EU EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is wuyimumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is wuyimumab.

Table 14; amino acid sequence of a 4-1BB agonist antibody associated with Wu Tumu mab.

In some embodiments, the 4-1BB agonist is monoclonal antibody Wu Ruilu mab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biological analog thereof. Wu Ruilu mab is available from Bai-Shi Miq precious corporation and Creative Biolabs, inc. Wu Ruilu monoclonal antibody is immunoglobulin G4-kappa antibody [ Chile TNFRSF9 (tumor necrosis factor receptor superfamily member 9,4-1BB, T cell antigen ILA, CD 137) ] Chile (fully human) monoclonal antibody. The amino acid sequence of Wu Ruilu mab is set forth in table 15. Wu Ruilu the mab comprises an N-glycosylation site at position 298 (and 298'); intra-heavy chain disulfide bonds at positions 22-95 (V _H-V_L)、148-204(C_H1-CL)、262-322(C_H 2) and 368-426 (C _H 3) (and at positions 22"-95", 148"-204", 262"-322" and 368 "-426"); light chain intra-chain disulfide bonds at positions 23'-88' (V _H-V_L) and 136'-196' (C _H 1-CL) (and at positions 23 '"-88'" and 136 '"-196'"); inter-chain heavy chain-heavy chain disulfide bonds at positions 227-227 "and 230-230"; and interchain heavy-light chain disulfide bonds at 135-216 'and 135 "-216'". The preparation and nature of Wu Ruilu mab and variants and fragments thereof are described in U.S. patent nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical characterization of Wu Ruilu mab is described in Segal et al, (Clin. Cancer Res.) 2016, please access http:/dx. Doi. Org/10.1158/1078-0432.CCR-16-1272. Current clinical trials of Wu Ruilu mab in a variety of blood and solid tumor indications include the national institutes of health, clinicaltrias, gov, identification numbers NCT01775631, NCT02110082, NCT02253992, and NCT01471210.

In some embodiments, the 4-1BB agonist comprises the sequence of SEQ ID NO:52 and the heavy chain shown in SEQ ID NO: 53. In some embodiments, the 4-1BB agonist comprises a sequence having the amino acid sequence of SEQ ID NO:52 and SEQ ID NO:53, or an antigen binding fragment, fab fragment, single chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 99% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 98% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 97% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 96% identity. In some embodiments, the 4-1BB agonist comprises a sequence corresponding to SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 95% identity.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of Wu Ruilu mab. In some embodiments, the 4-1BB agonist heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:54, and the 4-1BB agonist light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:55, and conservative amino acid substitutions thereof. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:54 and SEQ ID NO:55 has at least 99% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:54 and SEQ ID NO:55 has at least 98% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:54 and SEQ ID NO:55 has at least 97% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:54 and SEQ ID NO:55 has at least 96% identity. In some embodiments, the 4-1BB agonist comprises V _H and V _L regions, each of which hybridizes to SEQ ID NO:54 and SEQ ID NO:55 has at least 95% identity. In some embodiments, the 4-1BB agonist comprises an scFv antibody comprising the V _H and V _L regions, each of which hybridizes to the sequence set forth in SEQ ID NO:54 and SEQ ID NO:55 has at least 99% identity.

In some embodiments, the 4-1BB agonist comprises a sequence having the amino acid sequence of SEQ ID NO: 56. SEQ ID NO:57 and SEQ ID NO:58 and the heavy chain CDR1, CDR2 and CDR3 domains thereof with conservative amino acid substitutions; having the sequence of SEQ ID NO: 59. SEQ ID NO:60 and SEQ ID NO:61 and conservative amino acid substitutions thereof.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by a drug administration reference to Zuccinimide. In some embodiments, a biosimilar monoclonal antibody comprises a 4-1BB antibody that comprises an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product that comprises one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. The 4-1BB agonist antibodies may be obtained from a drug administration, such as U.S. FDA and/or EU EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is nivolumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is nivolumab.

Table 15: amino acid sequence of a 4-1BB agonist antibody associated with Wu Ruilu mab.

In some embodiments, the 4-1BB agonist is selected from the group consisting of: 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Siemens Feichi MS621 PABX), 145501 (Leinco Technologies B591), antibodies produced by cell lines maintained as ATCC No. HB-11248 and disclosed in U.S. Pat. No. 6,974,863, 5F4 (BioLegend 31 1503), C65-485 (BD Pharmingen 559446), antibodies disclosed in U.S. patent application publication No. US2005/0095244 (e.g., 20H4.9-IgGl (BMS-663031)), antibodies disclosed in U.S. Pat. No. 6,887,673 (e.g., 4E9 or BMS-554271), antibodies disclosed in U.S. Pat. No. 6,303,121, antibodies disclosed in U.S. Pat. No. 6,569,997, antibodies disclosed in U.S. Pat. No. 6,905,685 (e.S. 4E9 or BMS. 5236), antibodies disclosed in U.S. Pat. No. 5H 20H4.9-IgGl (e.S. 20) or antibodies disclosed in U.S. Pat. No. 5E 9-bars (e.S. 4E9 or BMS. 4635), antibodies disclosed in U.S. Pat. No. 5 A. 4E 3 or in U.S. Pat. No. 3B-3 or in U.S. Pat. No. 4695 (e.S. 4695); antibodies disclosed in U.S. patent No. 6,210,669 (e.g., 1D8, 3B8, or 3 El), antibodies described in U.S. patent No. 5,928,893, antibodies disclosed in U.S. patent No. 6,303,121, antibodies disclosed in U.S. patent No. 6,569,997, antibodies disclosed in international patent application publications nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, and fragments, derivatives, conjugates, variants, or biological analogs thereof, wherein the disclosure of each of the foregoing patents or patent application publications is incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonism fusion protein as described in: international patent application publications WO 2008/025516 A1, WO 2009/0071120 A1, WO 2010/003766 A1, WO 2010/010051A1 and WO 2010/078966 A1; U.S. patent application publication Nos. US2011/0027218A1, US 2015/0126209 A1, US 2011/011494 A1, US2015/0110734 A1 and US 2015/0126110 A1; and U.S. patent nos. 9,359,420, 9,340,599,8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein as depicted in structure I-A (C-terminal Fc antibody fragment fusion protein) or structure I-B (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biological analog thereof (see FIG. 18). In structures I-A and I-B, cylinders refer to individual polypeptide binding domains. Constructs I-A and I-B comprise three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL (4-1 BB ligand, CD137 ligand (CD 137L) or an antibody that binds 4-1BB, which TNFRSF binding domains fold to form a trivalent protein, which trivalent protein is then linked to a second trivalent protein by an IgG1-Fc (comprising C _H 3 and C _H 2 domains) which IgG1-Fc is then used to link the two trivalent proteins together by disulfide bonds (elongated small ellipses) to stabilize the structure and provide agonists capable of bringing together the intracellular signaling domains of the six receptors and signaling proteins to form a signaling complex; or references incorporated elsewhere herein, the fusion protein structures of this form are described in U.S. Pat. nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of structure I-A shown in FIG. 18 are shown in Table 16. The Fc domain preferably comprises the complete constant domain (amino acids 17-230 of SEQ ID NO: 62), the complete hinge domain (amino acids 1-16 of SEQ ID NO: 62) or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). Preferred linkers for linking the C-terminal Fc antibodies may be selected from the group consisting of SEQ ID NOs: 63 to SEQ ID NO:72, including linkers suitable for fusion of other polypeptides.

Table 16: amino acid sequence of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) having C-terminal Fc-antibody fragment fusion protein design (Structure I-A)

The amino acid sequences of the other polypeptide domains of structure I-B given in FIG. 18 are shown in Table 17. If the Fc antibody fragment is fused to the N-terminus of TNRFSF fusion proteins as in structure I-B, the sequence of the Fc module is preferably SEQ ID NO:73, the linker sequence is preferably selected from the group consisting of SED ID NO:74 to SEQ ID NO: 76.

Table 17: amino acid sequence of TNFRSF agonist fusion proteins (including 4-1BB agonist fusion proteins) having an N-terminal Fc-antibody fragment fusion protein design (structure I-B).

In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains selected from the group consisting of: wu Tumu variable heavy and variable light chains of mab, wu Ruilu variable heavy and variable light chains of mab, wu Tumu variable heavy and variable light chains of mab, variable heavy and variable light chains selected from the variable heavy and variable light chains described in table 18, any combination of the foregoing variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biological analogs thereof.

In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain containing a 4-1BBL sequence. In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more amino acid sequences according to SEQ ID NO:77, and a 4-1BB binding domain of the sequence of 77. In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain comprising a soluble 4-1BBL sequence. In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more amino acid sequences according to SEQ ID NO:78, and a 4-1BB binding domain of the sequence of 78.

In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains, which one or more 4-1BB binding domains are a polypeptide comprising a sequence that hybridizes to a sequence of SEQ ID NO:44 and SEQ id no:45, the V _H and V _L domains of the V _H and V _L domains having at least 95% identity are joined by a linker. In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains, which one or more 4-1BB binding domains are a polypeptide comprising a sequence that hybridizes to a sequence of SEQ ID NO:54 and SEQ ID NO:55, and the V _H and V _L domains of V _H and V _L domains having at least 95% identity are joined by a linker. In some embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains, which one or more 4-1BB binding domains are scFv domains comprising V _H and V _L regions each having at least 95% identity to the V _H and V _L sequences set forth in Table 18, the V _H and V _L domains being joined by a linker.

Table 18: other polypeptide domains suitable for use as 4-1BB binding domains in fusion proteins or as scFv 4-1BB agonist antibodies

In some embodiments, the 4-1BB agonist is a 4-1BB agonizing single chain fusion polypeptide, which comprises: (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminus and/or the C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In some embodiments, the 4-1BB agonist is a 4-1BB agonizing single chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain, wherein each soluble 4-1BB domain lacks a stem region (which facilitates trimerization and provides a distance from the cell membrane, but is not part of the 4-1BB binding domain), the first and second peptide linkers independently having a length of 3 to 8 amino acids.

In some embodiments, the 4-1BB agonist is a 4-1BB agonizing single chain fusion polypeptide comprising (i) a first soluble Tumor Necrosis Factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each soluble TNF superfamily cytokine domain lacks a stem region and the first and second peptide linkers independently have a length of 3 to 8 amino acids, each TNF superfamily cytokine domain being a 4-1BB binding domain.

In some embodiments, the 4-1BB agonist is a 4-1BB agonizing scFv antibody, which comprises any of the foregoing V _H domains linked to any of the foregoing V _L domains.

In some embodiments, the 4-1BB agonist is a BPS Bioscience 4-1BB agonist antibody, catalog number 79097-2, available from BPS Bioscience of san Diego, calif., U.S.A. In some embodiments, the 4-1BB agonist is a Creative Biolabs 4-1BB agonist antibody, catalog number MOM-18179, commercially available from Creative Biolabs of Xueli, N.Y..

OX40 (CD 134) agonist

In some embodiments, the TNFRSF agonist is an OX40 (CD 134) agonist. The OX40 agonist may be any OX40 binding molecule known in the art. The OX40 binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX 40. OX40 agonists or OX40 binding molecules may comprise immunoglobulin heavy chains of any isotype (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subclass of immunoglobulin molecule. OX40 agonists or OX40 binding molecules may have a heavy chain and a light chain. As used herein, the term binding molecule also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized or chimeric antibodies, and antibody fragments, such as Fab fragments, F (ab') fragments, fragments produced from a Fab expression library, engineered versions of any of the above epitope-binding fragments, and antibodies that bind to OX40, such as scFv molecules. In some embodiments, the OX40 agonist is an antigen binding protein of a fully human antibody. In some embodiments, the OX40 agonist is an antigen binding protein of a humanized antibody. In some embodiments, OX40 agonists useful in the methods and compositions of the present disclosure include anti-OX 40 antibodies, human anti-OX 40 antibodies, mouse anti-OX 40 antibodies, mammalian anti-OX 40 antibodies, monoclonal anti-OX 40 antibodies, polyclonal anti-OX 40 antibodies, chimeric anti-OX 40 antibodies, anti-OX 40 adnetant, anti-OX 40 domain antibodies, single chain anti-OX 40 fragments, heavy chain anti-OX 40 fragments, light chain anti-OX 40 fragments, anti-OX 40 fusion proteins, and fragments, derivatives, conjugates, variants, or biological analogs thereof. In some embodiments, the OX40 agonist is an agonistic anti-OX 40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

In some embodiments, the OX40 agonist or OX40 binding molecule may also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun et al, journal of immunotherapy 2009,182,1481-89. In some embodiments, a multimeric OX40 agonist, such as a trimeric or hexameric OX40 agonist (having three or six ligand binding domains), may induce superior receptor (OX 40L) clustering and internal cell signaling complex formation compared to an agonistic monoclonal antibody that typically has two ligand binding domains. Trimeric (trivalent) or hexameric (or hexavalent) more fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins are described, for example, in Gieffers et al, molecular cancer therapeutics 2013,12,2735-47.

Agonistic OX40 antibodies and fusion proteins are known to induce strong immune responses. Curti et al, cancer research 2013,73,7189-98. In some embodiments, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to an OX40 antigen in a manner that sufficiently reduces toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abrogates antibody-dependent cellular cytotoxicity (ADCC), e.g., NK cell cytotoxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates Antibody Dependent Cell Phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates Complement Dependent Cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abrogates Fc region function.

In some embodiments, the OX40 agonist is characterized as binding to human OX40 (SEQ ID NO: 85) with high affinity and agonistic activity. In some embodiments, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 85). In some embodiments, the OX40 agonist is a binding molecule that binds to murine OX40 (SEQ ID NO: 86). The amino acid sequences of OX40 antigens bound to OX40 agonists or binding molecules are summarized in table 19.

Table 19: amino acid sequence of OX40 antigen

In some embodiments, the described compositions, processes, and methods include an OX40 agonist that binds to human or murine OX40 at a K _D of about 100pM or less, to human or murine OX40 at a K _D of about 90pM or less, to human or murine OX40 at a K _D of about 80pM or less, to human or murine OX40 at a K _D of about 70pM or less, to human or murine OX40 at a K _D of about 60pM or less, to human or murine OX40 at a K _D of about 50pM or less, to human or murine OX40 at a K _D of about 40pM or to human or murine OX40 at a K _D of about 30pM or less.

In some embodiments, the described compositions, processes, and methods include an OX40 agonist that binds to human or murine OX40 at a k _assoc of about 7.5×10 ⁵ 1/m·s or faster, binds to human or murine OX40 at a k _assoc of about 7.5×10 ⁵ 1/m·s or faster, binds to human or murine OX40 at a k _assoc of about 8×10 ⁵ 1/m·s or faster, binds to human or murine OX40 at a k _assoc of about 8.5×10 ⁵ 1/m·s or faster, binds to human or murine OX40 at a k _assoc of about 9×10 ⁵ 1/m·s or faster, binds to human or murine OX40 at a k _assoc of about 9.5×10 ⁵ 1/m·s or faster, or binds to human or murine OX40 at a k _assoc of about 1×10 ⁶/m·s or faster.

In some embodiments, the described compositions, processes, and methods include an OX40 agonist that binds to human or murine OX40 at about 2 x 10 ^-5 1/s or slower k _dissoc, to human or murine OX40 at about 2.1 x 10 ^-5/s or slower k _dissoc, to human or murine OX40 at about 2.2 x 10 ^-5 1/s or slower k _dissoc, to human or murine OX40 at about 2.3 x 10 ^-5 1/s or slower k _dissoc, to human or murine OX40 at about 2.4 x 10 ^-5/s or slower k _dissoc, to human or murine OX40 at about 2.5 x 10 ^-5 1/s or slower k _dissoc/s, to human or murine OX40 at about 2.6 x 10 ^-5/s or slower k _dissoc, to human or murine OX40 at about 2.7 x 10 x 69/s or slower k _dissoc, to human or murine OX40 at about 2.4 x 10 k ^-5/s or slower k _dissoc, to human or murine OX40 at about 2.5 x 10 x 36/s or slower k 3793, to human or murine OX40 at about 2.6 x 10 x 36/s or slower k 3493/s or slower k 35.

In some embodiments, the described compositions, processes, and methods include an OX40 agonist that binds to human or murine OX40 with an IC ₅₀ of about 10nM or less, to human or murine OX40 with an IC ₅₀ of about 9nM or less, to human or murine OX40 with an IC ₅₀ of about 8nM or less, to human or murine OX40 with an IC ₅₀ of about 7nM or less, to human or murine OX40 with an IC ₅₀ of about 6nM or less, to human or murine OX40 with an IC ₅₀ of about 5nM or less, to human or murine OX40 with an IC ₅₀ of about 4nM or less, to human or murine OX40 with an IC ₅₀ of about 3nM or less, to human or murine OX40 with an IC ₅₀ of about 2nM or to human or murine OX40 with an IC ₅₀ of about 1nM or less.

In some embodiments, the OX40 agonist is a tower Wo Xishan antibody, also known as MEDI0562 or MEDI-0562. Tawoximab is available from the medical immune (MedImmune) subsidiary of the company Aspirikang (AstraZeneca, inc.). Tawacemide is an immunoglobulin G1-kappa anti [ Chile TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD 134) ] humanized and chimeric monoclonal antibody. The amino acid sequence of tavorax is set forth in table 20. Tawovens monoclonal antibodies comprise N-glycosylation sites at positions 301 and 301", with fucosylation complex biantennary CHO-type glycans; intra-heavy chain disulfide bonds at positions 22-95 (V _H-V_L)、148-204((C_H1-C_L)、265-325(C_H 2) and 371-429 (C _H) (and at positions 22"-95", 148"-204", 265"-325" and 371 "-429"); an intra-light chain disulfide bond at positions 23'-88' (V _H-V_L) and 134'-194' (C _H1-C_L) (and at positions 23 '"-88'" and 134 '"-194'"; interchain heavy chain-heavy chain disulfide bonds at positions 230-230 "and 233-233"; and interchain heavy-light chain disulfide bonds at 224-214 'and 224 "-214'". Current clinical trials of tavorax in various solid tumor indications include the national institutes of health, clinicaltrias identification numbers NCT02318394 and NCT02705482.

In some embodiments, the OX40 agonist comprises the amino acid sequence of SEQ ID NO:87 and the heavy chain shown in SEQ ID NO: 88. In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO:87 and SEQ ID NO:88, or an antigen binding fragment, fab fragment, single chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 99% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 98% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 97% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 96% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 95% identity.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of tavorexant. In some embodiments, OX40 agonist heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:89, and the OX40 agonist light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:90, and conservative amino acid substitutions thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:89 and SEQ ID NO:90 has V _H and V _L regions of at least 99% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:89 and SEQ ID NO:90 has V _H and V _L regions of at least 98% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:89 and SEQ ID NO:90 has V _H and V _L regions of at least 97% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:89 and SEQ ID NO:90 has V _H and V _L regions of at least 96% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:89 and SEQ ID NO:90 has V _H and V _L regions of at least 95% identity. In some embodiments, the OX40 agonist comprises an scFv antibody comprising an amino acid sequence that is each identical to SEQ ID NO:89 and SEQ ID NO:90 has V _H and V _L regions of at least 99% identity.

In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO: 91. SEQ ID NO:92 and SEQ ID NO:93 and the heavy chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 94. SEQ ID NO:95 and SEQ ID NO:96 and conservative amino acid substitutions thereof.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug administration reference tawoxib. In some embodiments, a biological analog monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is tavortioximab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is tavorax. OX40 agonist antibodies may be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is tavorexant. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is tavorexant.

Table 20: amino acid sequence of an OX40 agonist antibody associated with tavorax.

In some embodiments, the OX40 agonist is 11D4, which is a fully human antibody available from the company pyroes. The preparation and characterization of 11D4 is described in U.S. patent nos. 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is set forth in table 21.

In some embodiments, the OX40 agonist comprises the amino acid sequence of SEQ ID NO:97 and the heavy chain shown in SEQ ID NO: 98. In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO:97 and SEQ ID NO:98, or antigen binding fragments, fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:97 and SEQ ID NO:98, and a heavy chain and a light chain having at least 99% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:97 and SEQ ID NO:98, and a heavy chain and a light chain having at least 98% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:97 and SEQ ID NO:98, and a heavy chain and a light chain having at least 97% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:97 and SEQ ID NO:98, and a heavy chain and a light chain having at least 96% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:97 and SEQ ID NO:98, and a heavy chain and a light chain having at least 95% identity.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of 11D 4. In some embodiments, OX40 agonist heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:99, the OX40 agonist light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:100, and conservative amino acid substitutions thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:99 and SEQ ID NO:100 has V _H and V _L regions of at least 99% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:99 and SEQ ID NO:100 has V _H and V _L regions of at least 98% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:99 and SEQ ID NO:100 has V _H and V _L regions of at least 97% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:99 and SEQ ID NO:100 has V _H and V _L regions of at least 96% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:99 and SEQ ID NO:100 has V _H and V _L regions of at least 95% identity.

In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO: 101. SEQ ID NO:102 and SEQ ID NO:103 and the heavy chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 104. SEQ ID NO:105 and SEQ ID NO:106 and conservative amino acid substitutions thereof.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody that is approved by a drug administration with reference to 11D4. In some embodiments, a biological analog monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an OX40 agonist antibody that is licensed or otherwise subject to authorization, wherein the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is 11D4.OX40 agonist antibodies may be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is 11D4. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is 11D4.

Table 21: amino acid sequence of 11D 4-related OX40 agonist antibody

In some embodiments, the OX40 agonist is 18D8, which is a fully human antibody available from the company pyroes. The preparation and characterization of 18D8 is described in U.S. patent nos. 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is set forth in table 22.

In some embodiments, the OX40 agonist comprises the amino acid sequence of SEQ ID NO:107 and the heavy chain shown in SEQ ID NO: 108. In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO:107 and SEQ ID NO:108, or an antigen binding fragment, fab fragment, single chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:107 and SEQ ID NO:108 has at least 99% identity to the heavy and light chains. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:107 and SEQ ID NO:108 has at least 98% identity to the heavy and light chains. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:107 and SEQ ID NO:108 has at least 97% identity to the heavy and light chains. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:107 and SEQ ID NO:108 has at least 96% identity to the heavy and light chains. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:107 and SEQ ID NO:108 has at least 95% identity to the heavy and light chains.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of 18D 8. In some embodiments, OX40 agonist heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:109, the OX40 agonist light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:110, and conservative amino acid substitutions thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:109 and SEQ ID NO:110, and V _H and V _L regions having at least 99% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:109 and SEQ ID NO:110, and V _H and V _L regions having at least 98% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:109 and SEQ ID NO:110, and V _H and V _L regions having at least 97% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:109 and SEQ ID NO:110 has V _H and V _L regions of at least 96% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:109 and SEQ ID NO:110, and V _H and V _L regions of at least 95% identity.

In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO: 111. SEQ ID NO:112 and SEQ ID NO:113 and the heavy chain CDR1, CDR2, and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 114. SEQ ID NO:115 and SEQ ID NO:116 and the light chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug administration with reference to 18D8. In some embodiments, a biological analog monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is 18D8.OX40 agonist antibodies may be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is 18D8. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is 18D8.

Table 22: amino acid sequence of an OX40 agonist antibody associated with 18D 8.

In some embodiments, the OX40 agonist is Hu119-122, which is a humanized antibody available from glazin smith public limited (GlaxoSmithKline plc). The preparation and properties of Hu119-122 are described in U.S. patent nos. 9,006,399 and 9,163,085 and international patent publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequences of Hu119-122 are set forth in Table 23.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VR) of Hu 119-122. In some embodiments, OX40 agonist heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:117, the OX40 agonist light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:118 and conservative amino acid substitutions thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:117 and SEQ ID NO:118 has V _H and V _L regions of at least 99% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:117 and SEQ ID NO:118 has V _H and V _L regions of at least 98% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:117 and SEQ ID NO:118 has V _H and V _L regions of at least 97% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:117 and SEQ ID NO:118 has V _H and V _L regions of at least 96% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:117 and SEQ ID NO:118 has V _H and V _L regions of at least 95% identity.

In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO: 119. SEQ ID NO:120 and SEQ ID NO:121 and the heavy chain CDR1, CDR2, and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 122. SEQ ID NO:123 and SEQ ID NO:124 and the light chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug administration with reference to Hu119-122. In some embodiments, a biological analog monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122.OX40 agonist antibodies may be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is Hu119-122.

Table 23: amino acid sequence of an OX40 agonist antibody associated with Hu 119-122.

In some embodiments, the OX40 agonist is Hu106-222, which is a humanized antibody available from glazin smith co-ltd. The preparation and properties of Hu106-222 are described in U.S. patent nos. 9,006,399 and 9,163,085 and international patent publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequences of Hu106-222 are set forth in Table 24.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VR) of Hu 106-222. In some embodiments, OX40 agonist heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:125, the OX40 agonist light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:126, and conservative amino acid substitutions thereof. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:125 and SEQ ID NO:126 has V _H and V _L regions of at least 99% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:125 and SEQ ID NO:126 has V _H and V _L regions of at least 98% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:125 and SEQ ID NO:126 has V _H and V _L regions of at least 97% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:125 and SEQ ID NO:126 has V _H and V _L regions of at least 96% identity. In some embodiments, the OX40 agonist comprises an amino acid sequence that is each identical to SEQ ID NO:125 and SEQ ID NO:126 has V _H and V _L regions of at least 95% identity.

In some embodiments, the OX40 agonist comprises an amino acid sequence having SEQ ID NO: 127. SEQ ID NO:128 and SEQ ID NO:129 and the heavy chain CDR1, CDR2, and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 130. SEQ ID NO:131 and SEQ ID NO:132 and the light chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug administration with reference to Hu106-222. In some embodiments, a biological analog monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222.OX40 agonist antibodies may be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is Hu106-222.

Table 24: amino acid sequence of an OX40 agonist antibody associated with Hu 106-222.

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B 12). MEDI6469 is a murine monoclonal antibody. Weinberg et al, J.Immunotherapy, 2006,29,575-585. In some embodiments, the OX40 agonist is an antibody produced by the 9B12 hybridoma, saved by Biovest inc (Malvern, MA, USA), as described in Weinberg et al, journal of immunotherapy, 2006,29,575-585, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises CDR sequences of MEDI 6469. In some embodiments, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of MEDI 6469.

In some embodiments, the OX40 agonist is L106 BD (Pharmingen, product number 340420). In some embodiments, the OX40 agonist comprises the CDRs of antibody L106 (BD Pharmingen, product No. 340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106 (BD Pharmingen, product No. 340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, cat No. 20073). In some embodiments, the OX40 agonist comprises the CDRs of antibody ACT35 (Santa Cruz Biotechnology, cat# 20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, cat# 20073). In some embodiments, the OX40 agonist is the murine monoclonal antibody anti-mCD 134/mxx 40 (clone OX 86), commercially available from InVivoMAb of BioXcell Inc of parisons, new hampshire.

In some embodiments, the OX40 agonist is selected from the OX40 agonists described in the following: international patent application publications WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191 and WO 2014/148895; european patent application EP 0672141; U.S. patent application publication nos. US2010/136030, US2014/377284, US 2015/190506 and US 2015/132088 (including clone 20E5 and 12H 3); and U.S. patent nos. 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of each of which are incorporated herein by reference in their entirety.

In some embodiments, the OX40 agonist is an OX40 agonist fusion protein as depicted in structure I-a (C-terminal Fc-antibody fragment fusion protein) or structure I-B (N-terminal Fc-antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biological analog thereof. The characteristics of structures I-A and I-B have been described above and in U.S. Pat. Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequences of the polypeptide domains of structure I-A shown in FIG. 18 are shown in Table 16. The Fc domain preferably comprises the complete constant domain (amino acids 17-230 of SEQ ID NO: 62), the complete hinge domain (amino acids 1-16 of SEQ ID NO: 62) or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 62). Preferred linkers for linking the C-terminal Fc antibodies may be selected from the group consisting of SEQ ID NOs: 63 to SEQ ID NO:72, including linkers suitable for fusion of other polypeptides. Also, the amino acid sequences of the polypeptide domains of structure I-B are given in FIG. 18 are shown in Table 17. If the Fc antibody fragment is fused to the N-terminus of TNRFSF fusion proteins as in structure I-B, the sequence of the Fc module is preferably SEQ ID NO:73, and the linker sequence is preferably selected from the group consisting of SED ID NO:74 to SEQ ID NO: 76.

In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains selected from the group consisting of: the variable heavy and variable light chains of tavacizumab, the variable heavy and variable light chains of 11D4, the variable heavy and variable light chains of 18D8, the variable heavy and variable light chains of Hu119-122, the variable heavy and variable light chains of Hu106-222, the variable heavy and variable light chains selected from the variable heavy and variable light chains described in table 25, any combination of the foregoing, and fragments, derivatives, conjugates, variants, and biological analogs thereof.

In some embodiments, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain comprising an OX40L sequence. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more polypeptides comprising an amino acid sequence according to SEQ ID NO:133, an OX40 binding domain of the sequence of 133. In some embodiments, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain comprising a soluble OX40L sequence. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more polypeptides comprising an amino acid sequence according to SEQ ID NO:134, and an OX40 binding domain of the sequence of 134. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more polypeptides comprising an amino acid sequence according to SEQ ID NO:135, and an OX40 binding domain of the sequence of 135.

In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains that are scFv domains comprising a sequence that is each identical to SEQ ID NO:89 and SEQ ID NO:90, wherein the V _H and V _L domains of V _H and V _L are joined by a linker. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains that are scFv domains comprising a sequence that is each identical to SEQ ID NO:99 and SEQ ID NO:100, wherein the V _H and V _L domains of V _H and V _L are joined by a linker. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains that are scFv domains comprising a sequence that is each identical to SEQ ID NO:109 and SEQ ID NO:110, wherein the V _H and V _L domains are joined by a linker. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains that are scFv domains comprising a sequence that is each identical to SEQ ID NO:127 and SEQ ID NO:128, wherein the V _H and V _L domains of V _H and V _L are joined by a linker. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains that are scFv domains comprising a sequence that is each identical to SEQ ID NO:125 and SEQ ID NO:126, wherein the V _H and V _L domains are joined by a linker. In some embodiments, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains that are scFv domains comprising V _H and V _L regions each having at least 95% identity to V _H and V _L sequences provided in table 25, wherein the V _H and V _L domains are linked by a linker.

Table 25: OX40 binding domains suitable for use as fusion proteins (e.g., structures I-a and I-B) or other polypeptide domains suitable for use as scFv OX40 agonist antibodies

In some embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain further comprising an additional domain at the N-terminus and/or the C-terminus, and wherein the additional domain is a Fab or Fc fragment domain. In some embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain further comprising an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain, wherein each soluble OX40 binding domain lacks a stem region (which promotes trimerization and provides a certain distance from the cell membrane, but is not part of the OX40 binding domain), the first and second peptide linkers independently having a length of 3 to 8 amino acids.

In some embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising: (i) A first soluble Tumor Necrosis Factor (TNF) superfamily cytokine domain; (ii) a first peptide linker; (iii) a second soluble TNF superfamily cytokine domain; (iv) a second peptide linker; and (v) a third soluble TNF superfamily cytokine subdomain, wherein each soluble TNF superfamily cytokine domain lacks a stem region, the first and second peptide linkers independently have a length of 3 to 8 amino acids, wherein the TNF superfamily cytokine domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383.MEDI6383 is an OX40 agonist fusion protein and may be prepared as described in U.S. patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In some embodiments, the OX40 agonist is an OX40 agonist scFv antibody comprising any one of the foregoing V _H domains linked to any one of the foregoing V _L domains.

In some embodiments, the OX40 agonist is Creative Biolabs OX agonist monoclonal antibody MOM-18555, available from Creative Biolabs, inc.

In some embodiments, the OX40 agonist is the OX40 agonist antibody clone bor-ACT 35, available from BioLegend, inc.

B. alternative cell viability assay

Alternatively, after initiating the first amplification (sometimes referred to as initial subject amplification (initial bulk expansion)), a cell viability assay may be performed using standard assays known in the art. Thus, in certain embodiments, the method comprises performing a cell viability assay after initiating the first amplification. For example, a trypan blue exclusion analysis may be performed on a subject TIL sample that selectively marks dead cells and allows for viability assessment. Other assays for testing survival may include, but are not limited to, alamarblue (alamarblue) assays and MTT assays.

1. Cell count, viability, flow cytometry

In some embodiments, cell count and/or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56, and any other markers disclosed or described herein, can be measured by flow cytometry using a FACSCanto ^TM flow cytometer (BD Biosciences), with antibodies such as, but not limited to, those available from the bi bioscience (bi Biosciences, san jose, california). The cells may be manually calculated using a single-use c-chip hemocytometer (VWR, badavia, il) and viability assessed using any method known in the art, including but not limited to trypan blue staining. Cell viability may also be analyzed based on U.S. patent application publication No. 2018/0282694, which is incorporated herein by reference in its entirety. Cell viability may also be analyzed based on U.S. patent application publication No. 2018/0280436 or international patent application publication No. WO/2018/081473, which are incorporated herein by reference in their entirety for all purposes.

In some cases, the subject TIL population may be immediately cryopreserved using the protocol discussed below. Alternatively, the subject TIL population may be REP-subjected and then cryopreserved as described below. Similarly, in cases where a genetically modified TIL is to be used in therapy, the subject or population of REP TILs may be genetically modified for appropriate treatment.

2. Cell culture

In some embodiments, methods for amplifying TIL (including those described above and illustrated in fig. 1 and 8, particularly, e.g., in fig. 8A and/or 8B and/or 8C and/or 8D) may include using about 5,000ml to about 25,000ml of cell culture medium, about 5,000ml to about 10,000ml of cell culture medium, or about 5,800ml to about 8,700ml of cell culture medium. In some embodiments, the medium is serum-free medium. In some embodiments, the medium in the initial first amplification is serum-free. In some embodiments, the medium in the second amplification is serum-free. In some embodiments, the medium in both the initial first amplification and the second amplification (also referred to as the rapid second amplification) is serum-free. In some embodiments, no more than one type of cell culture medium is used to amplify the number of TILs. Any suitable cell culture medium may be used, such as AIM-V cell culture medium (L-glutamine, 50. Mu.M streptomycin sulfate, and 10. Mu.M gentamicin sulfate) cell culture medium (Injegen, inc. (Invitrogen)), in which case the methods of the present invention advantageously reduce the amount of culture medium and the number of culture medium types required to amplify the number of TILs.

In some embodiments, the cell culture medium in the first and/or second gas permeable containers is unfiltered. The use of unfiltered cell culture media can simplify the procedure required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks β -mercaptoethanol (BME).

In some embodiments, the method comprises obtaining a tumor tissue sample from a mammal during the method; culturing a tumor tissue sample in a first gas-permeable container containing a cell culture medium comprising IL-2, 1X antigen presenting feeder cells, and OKT-3 for a period of about 1 to 8 days, e.g., about 7 days as the initiation of first expansion and about 8 days as the initiation of first expansion; transferring the TIL to a second gas permeable container containing a cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3 and amplifying the number of TILs in the second gas permeable container for a period of about 7 to 9 days (e.g., about 7 days, about 8 days, or about 9 days).

In some embodiments, the method comprises obtaining a tumor tissue sample from a mammal during the method; culturing a tumor tissue sample in a first gas-permeable container containing a cell culture medium comprising IL-2, 1X antigen presenting feeder cells, and OKT-3 for a period of about 1 to 7 days (e.g., about 7 days) as an initiation of the first expansion; transferring the TIL to a second permeable container containing a cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3 and amplifying the number of TILs in the second permeable container for a period of about 7 to 14 days or about 7 to 9 days (e.g., about 7 days, about 8 days, about 9 days, about 10 days, or about 11 days).

In some embodiments, the method comprises obtaining a tumor tissue sample from a mammal during the method; culturing a tumor tissue sample in a first gas-permeable container containing a cell culture medium comprising IL-2, 1X antigen presenting feeder cells, and OKT-3 for a period of about 1 to 7 days (e.g., about 7 days) as an initiation of the first expansion; transferring the TIL to a second permeable container containing a cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3 and amplifying the number of TILs in the second permeable container for a period of time ranging from about 7 to 11 days (e.g., about 7 days, about 8 days, about 9 days, about 10 days, or about 11 days).

In some embodiments, the TIL is amplified in a gas-permeable container. TIL has been amplified using a gas permeable container, using PBMCs, using methods, compositions, and devices known in the art, including those described in U.S. patent application publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In some embodiments, the TIL is amplified in a gas permeable bag. In some embodiments, the TIL is amplified using a cell expansion system that expands the TIL in an air-permeable bag (e.g., xuri cell expansion system W25 (GE HEALTHCARE)). In some embodiments, the TIL is amplified using a cell expansion system that expands the TIL in an air-permeable bag (e.g., a WAVE bioreactor system, also referred to as Xuri cell expansion system W5 (GE HEALTHCARE)). In some embodiments, the cell expansion system comprises a gas permeable cell bag having a volume selected from the group consisting of: about 100mL, about 200mL, about 300mL, about 400mL, about 500mL, about 600mL, about 700mL, about 800mL, about 900mL, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, and about 10L.

In some embodiments, TIL may be amplified in a G-REX flask (available from Wilson wolf manufacturing Co.). Such embodiments allow cell populations to expand from about 5×10 ⁵ cells/cm ² to between 10×10 ⁶ and 30×10 ⁶ cells/cm ². In some embodiments, it is not fed. In some embodiments, it is not fed as long as the medium in the G-REX flask is at a height of about 10 cm. In some embodiments, it is not fed but more than one cytokine is added. In some embodiments, the cytokine may be added in a bolus, without mixing the cytokine with the medium. Such vessels, devices and methods are known in the art and have been used to amplify TIL, including those described in the following: U.S. patent application publication No. US2014/0377739A1, international publication No. WO 2014/210036 A1, U.S. patent application publication No. US 2013/0112017 A1, international publication No. WO 2013/188427 A1, U.S. patent application publication No. US 2011/0137128 A1, U.S. patent No. US 8,809,050 B2, international publication No. WO 2011/072088 A2, U.S. patent application publication No. US2016/0208216 A1, U.S. patent application publication No. US 2012/024133 A1, international publication No. WO 2012/129201 A1, U.S. patent application publication No. US 2013/2075 A1, U.S. patent No. 8,956,860 B2, international publication No. WO 2013/1735 A1, U.S. patent application publication No. US 2015/0175966A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al, J.Immunotherapy, 2012, 35:283-292.

Selectable gene knockdown or gene knockdown in TIL

In some embodiments, the amplified TILs of the present invention are further manipulated to transiently alter protein expression prior to, during, or after the amplification step, including during the closed aseptic manufacturing process (each as provided herein). In some embodiments, the transiently altered protein expression is due to transient gene editing. In some embodiments, the amplified TILs of the invention are treated with Transcription Factors (TF) and/or other molecules capable of transiently altering protein expression in the TILs. In some embodiments, the TF and/or other molecules capable of transiently altering protein expression provide for altered tumor antigen expression and/or altered number of tumor antigen specific T cells in the TIL population.

In certain embodiments, the method comprises gene editing the TIL population. In certain embodiments, the method comprises genetically editing the first population of TILs, the second population of TILs, and/or the third population of TILs.

In some embodiments, the invention includes gene editing by nucleotide insertion, such as by ribonucleic acid (RNA) insertion, including insertion of messenger RNA (mRNA) or small (or short) interfering RNAs (siRNA) into a TIL population to facilitate expression or inhibit expression of more than one protein and simultaneously facilitate the combination of one set of proteins with inhibiting another set of proteins.

In some embodiments, the amplified TILs of the present invention undergo transient changes in protein expression. In some embodiments, transiently altering protein expression occurs in a population of bulk TILs prior to the first amplification, including, for example, from a population of TILs obtained from step a, e.g., as indicated in fig. 8 (particularly fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, transiently altering protein expression occurs during the first amplification, including, for example, in a TIL population obtained from step B as indicated, for example, in fig. 8 (e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, transiently altering protein expression occurs after the first amplification, including, for example, a population of TILs that transitions between the first and second amplifications (e.g., a second population of TILs as described herein), a population of TILs obtained from step B, e.g., as indicated in fig. 8, and included in step C. In some embodiments, transiently altering protein expression is in the subject TIL population prior to the second amplification, including, for example, in a TIL population obtained from step C, e.g., as indicated in fig. 8, and prior to its amplification in step D. In some embodiments, transiently altering protein expression is during the second amplification, including, for example, in a TIL population (e.g., a third TIL population) amplified in step D, e.g., as indicated in fig. 8. In some embodiments, transiently altering protein expression occurs after the second amplification, including, for example, in a TIL population obtained from amplification in step D, e.g., as indicated in fig. 8.

In some embodiments, the method of transiently altering protein expression in a TIL population comprises the step of electroporation. Electroporation methods are known in the art and are described, for example, in the following: tsong J biophysics 1991,60,297-306 and U.S. patent application publication No. 2014/0227237A1, the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of transiently altering protein expression in a TIL population comprises the step of calcium phosphate transfection. Methods of calcium phosphate transfection (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in the following: graham and van der Eb, virology 1973,52,456-467; wigler et al, proc. Natl. Acad. Sci.) "1979,76,1373-1376; and Chen and Okayarea, molecular cell biology (mol. Cell. Biol.) 1987,7,2745-2752; and U.S. patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of transiently altering protein expression in a TIL population comprises a step of lipofection. Liposome transfection methods, for example, using 1:1 (w/w) liposome formulations of the cationic lipids N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) in filtered water are known in the art and are described in the following: rose et al, biotechnology (Biotechniques) 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA, 1987,84,7413-7417 and U.S. Pat. Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484 and 7,687,070, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of transiently altering protein expression in a TIL population comprises a transfection step using the method described in: U.S. patent nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of each of which are incorporated herein by reference.

In some embodiments, transiently altering protein expression results in an increase in memory stem T cells (TSCM). TSCM is an early precursor cell of an antigen that undergoes central memory T cells. TSCM generally exhibits the ability to define long-term survival, self-renewal, and multipotency of stem cells, and is generally required to produce effective TIL products. In a mouse model of adoptive cell transfer, TSCM has been shown to have enhanced antitumor activity compared to other T cell subsets. In some embodiments, transiently altering protein expression results in a TIL population having a composition comprising a high proportion of TSCM. In some embodiments, transiently altering protein expression results in an increase in the percentage of TSCM of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, transiently altering protein expression results in at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCM in the TIL population. In some embodiments, transiently altering protein expression results in a population of TILs having at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% tscm. In some embodiments, transiently altering protein expression results in a therapeutic TIL population having at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM.

In some embodiments, transiently altering protein expression results in the antigen undergoing T cell resuscitation (rejuvenation). In some embodiments, resuscitation includes, for example, increasing proliferation, increasing T cell activation, and/or increasing antigen recognition.

In some embodiments, transiently altering protein expression alters expression of a majority of T cells to preserve a tumor-derived TCR reservoir. In some embodiments, transiently altering protein expression does not alter the tumor-derived TCR reservoir. In some embodiments, transiently altering protein expression maintains a tumor-derived TCR reservoir.

In some embodiments, transiently altering a protein results in altering expression of a particular gene. In some embodiments, transiently altering protein expression targets includes (but is not limited to) the following genes: PD-1 (also known as PDCD1 or CC 279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, chimeric co-stimulatory receptor (CCR)、IL-2、IL-12、IL-15、IL-21、NOTCH 1/2ICD、CTLA-4、TIM3、LAG3、TIGIT、TET2、TGFβ、CCR2、CCR4、CCR5、CXCR1、CXCR2、CSCR3、CCL2(MCP-1)、CCL3(MIP-1α)、CCL4(MIP1-β)、CCL5(RANTES)、CXCL1/CXCL8、CCL22、CCL17、CXCL1/CXCL8、VHL、CD44、PIK3CD、SOCS1、 thymocyte selection-associated High Mobility Group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD 11), BCL6 co-inhibitor (BCOR), and/or cAMP Protein Kinase A (PKA). In some embodiments, transiently altering protein expression targets a gene selected from the group consisting of: PD-1, TGFBR2, CCR4/5, CTLA-4, CBLB (CBL-B), CISH, chimeric co-stimulatory receptor (CCR)、IL-2、IL-12、IL-15、IL-21、NOTCH 1/2ICD、TIM3、LAG3、TIGIT、TET2、TGFβ、CCR2、CCR4、CCR5、CXCR1、CXCR2、CSCR3、CCL2(MCP-1)、CCL3(MIP-1α)、CCL4(MIP1-β)、CCL5(RANTES)、CXCL1/CXCL8、CCL22、CCL17、CXCL1/CXCL8、VHL、CD44、PIK3CD、SOCS1、 thymocyte selection-related High Mobility Group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD 11), BCL6 co-inhibitor (BCOR) and/or cAMP Protein Kinase A (PKA). In some embodiments, the transient alteration of protein expression targets PD-1. In some embodiments, the transient alteration of protein expression targets TGFBR2. In some embodiments, the transient alteration of protein expression targets CCR4/5. In some embodiments, the transient alteration of protein expression targets CTLA-4. In some embodiments, the transient alteration of protein expression targets CBLB. In some embodiments, the transient alteration in protein expression targets CISH. In some embodiments, the transient alteration of protein expression targets CCR (chimeric co-stimulatory receptor). In some embodiments, transiently altering protein expression targets IL-2. In some embodiments, the transient change in protein expression targeting IL-12. In some embodiments, transiently altering protein expression targets IL-15. In some embodiments, transiently altering protein expression targets IL-21. In some embodiments, the transient altered protein expression targets NOTCH 1/2ICD. For some embodiments, transient alterations in protein expression target TIM3. In some embodiments, the transient alteration of protein expression targets LAG3. In some embodiments, the transient alteration of protein expression targets TIGIT. In some embodiments, the transient alteration of protein expression targets TET2. In some embodiments, the transient alteration of protein expression targets tgfβ. In some embodiments, the transient alteration of protein expression targets CCR1. In some embodiments, the transient alteration of protein expression targets CCR2. In some embodiments, the transient alteration of protein expression targets CCR4. In some embodiments, the transient alteration of protein expression targets CCR5. In some embodiments, transiently altering protein expression targets CXCR1. In some embodiments, transiently altering protein expression targets CXCR2. In some embodiments, the transient altered protein expression targets CSCR3. In some embodiments, the transient altered protein expression targets CCL2 (MCP-1). In some embodiments, the transient alteration of protein expression targets CCL3 (MIP-1α). In some embodiments, the transient alteration of protein expression targets CCL4 (MIP 1- β). In some embodiments, the transient altered protein expression targets CCL5 (RANTES). In some embodiments, the transient alteration of protein expression targets CXCL1. In some embodiments, the transient alteration of protein expression targets CXCL8. In some embodiments, the transient alteration of protein expression targets CCL22. In some embodiments, the transient alteration of protein expression targets CCL17. In some embodiments, the transient alteration of protein expression targets VHL. In some embodiments, the transient alteration of protein expression targets CD44. In some embodiments, the transient alteration of protein expression targets PIK3CD. In some embodiments, the transient alteration of protein expression targets SOCS1. In some embodiments, transient alterations in protein expression target thymocytes select a related High Mobility Group (HMG) box (TOX). In some embodiments, the transient alteration in protein expression targets ankyrin repeat domain 11 (ANKRD 11). In some embodiments, the transient alteration of protein expression targets a BCL6 co-inhibitor (BCOR). In some embodiments, the transient altered protein expression targets cAMP Protein Kinase A (PKA).

In some embodiments, transiently altering protein expression results in increased and/or over-expression of a chemokine receptor. In some embodiments, chemokine receptors that are overexpressed due to transient protein expression include receptors having ligands including, but not limited to, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP 1- β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.

In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1, CTLA-4, CBLB, CISH, TIM-3, LAG-3, TIGIT, TET2, TGF-beta R2, and/or TGF-beta (including resulting in, for example, a TGF-beta pathway block). In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CBLB (CBL-B). In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CISH. For some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIM-3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of LAG-3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIGIT. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TET 2. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of tgfβr2. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of tgfβ.

In some embodiments, transiently altering protein expression results in increased and/or over-expression of chemokine receptors, for example, to improve TIL transport or movement to a tumor site. In some embodiments, transiently altering protein expression results in increased and/or over-expression of a chimeric co-stimulatory receptor (CCR). In some embodiments, transiently altering protein expression results in increased and/or over-expression of a chemokine receptor selected from the group consisting of: CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2 and/or CSCR3..

In some embodiments, transiently altering protein expression results in increased and/or over-expression of interleukins. In some embodiments, transiently altering protein expression results in increased and/or over-expression of an interleukin selected from the group consisting of: IL-2, IL-12, IL-15, IL-18 and/or IL-21.

In some embodiments, transiently altering protein expression results in increased and/or over-expression of NOTCH 1/2 ICD. In some embodiments, transiently altering protein expression results in increased and/or over-expression of VHL. In some embodiments, transiently altering protein expression results in increased and/or over-expression of CD 44. In some embodiments, transiently altering protein expression results in increased and/or over-expression of PIK3 CD. In some embodiments, transiently altering protein expression results in increased and/or over-expression of SOCS 1.

In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of cAMP Protein Kinase A (PKA).

In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of a molecule selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGF beta R2, PKA, CBLB, BAFF (BR 3) and combinations thereof. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of two molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGF beta R2, PKA, CBLB, BAFF (BR 3) and combinations thereof. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and one molecule selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, tgfβr2, PKA, CBLB, BAFF (BR 3), and combinations thereof. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of: PD-1, CTLA-4, LAG-3, CISH, CBLB, TIM, TIGIT and combinations thereof. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and one of the following: CTLA-4, LAG3, CISH, CBLB, TIM, TIGIT, TET2, and combinations thereof. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and CTLA-4. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and LAG 3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and CISH. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and CBLB. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and TIM 3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and TIGIT. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of PD-1 and TET 2. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CTLA-4 and LAG 3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CTLA-4 and CISH. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CTLA-4 and CBLB. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CTLA-4 and TIM 3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CTLA-4 and TIGIT. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CTLA-4 and TET 2. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of LAG3 and CISH. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of LAG3 and CBLB. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of LAG3 and TIM 3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of LAG3 and TIGIT. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of LAG3 and TET 2. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CISH and CBLB. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CISH and TIM 3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CISH and TIGIT. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CISH and TET 2. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CBLB and TIM 3. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CBLB and TIGIT. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of CBLB and TET 2. For some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIM3 and PD-1. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIM3 and LAG 3. For some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIM3 and CISH. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIM3 and CBLB. For some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIM3 and TIGIT. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of TIM3 and TET 2.

In some embodiments, an adhesion molecule selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted into the first population of TILs, the second population of TILs, or the population of collected TILs by a gamma retrovirus or lentivirus method (e.g., expression of the adhesion molecule is increased).

In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, tgfβr2, PKA, CBLB, BAFF (BR 3), and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, transiently altering protein expression results in reduced and/or decreased expression of a molecule selected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, CISH, CBLB, TIGIT, TET2, and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, expression is reduced by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

In some embodiments, expression is increased by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some embodiments, expression is increased by at least about 90%. In some embodiments, expression is increased by at least about 95%. In some embodiments, expression is increased by at least about 99%.

In some embodiments, transiently altering protein expression is induced by treating the TIL with a Transcription Factor (TF) and/or other molecules capable of transiently altering protein expression in the TIL. In some embodiments, intracellular delivery of Transcription Factors (TF) and/or other molecules capable of transiently altering protein expression is performed using a microfluidic platform without an SQZ vector. Such methods demonstrate the ability to deliver proteins, including transcription factors, to a variety of primary human cells, including T cells, which have been described in U.S. patent application publication nos. US2019/0093073 A1, US2018/0201889 A1 and US 2019/0017072A1, the respective disclosures of which are incorporated herein by reference. Such methods may be used in the present invention to expose a TIL population to Transcription Factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression provide for increased expression of tumor antigens and/or increased numbers of tumor antigen-specific T cells in the TIL population, thereby resulting in increased efficacy of treatment of the TIL population to reprogram and reprogram the TIL population compared to a non-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in effector T cells and/or central memory T cell subpopulations relative to the starting or previous TIL population (i.e., prior to reprogramming), as described herein.

In some embodiments, transcription Factors (TF) include (but are not limited to) TCF-1, NOTCH 1/2ICD and/or MYB. In some embodiments, the Transcription Factor (TF) is TCF-1. In some embodiments, the Transcription Factor (TF) is NOTCH 1/2ICD. In some embodiments, the Transcription Factor (TF) is MYB. In some embodiments, the Transcription Factor (TF) is administered with an induced pluripotent stem cell culture (iPSC), such as a commercial KNOCKOUT serum replacement (Gibco/sameimer femto), to induce additional TIL reprogramming. In some embodiments, the Transcription Factor (TF) is administered with the iPSC mixture to induce additional TIL reprogramming. In some embodiments, the Transcription Factor (TF) is not administered with the iPSC mixture. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some embodiments, reprogramming results in a percentage increase in TSCM of about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% TSCM.

In some embodiments, the methods of transiently altering protein expression as described above may be combined with methods of genetically modifying a population of TILs, including the step of stably incorporating genes to produce more than one protein. In certain embodiments, the method comprises the step of genetically modifying the population of TILs. In certain embodiments, the method comprises genetically modifying the first population of TILs, the second population of TILs, and/or the third population of TILs. In some embodiments, the method of genetically modifying a population of TILs comprises the step of retroviral transduction. In some embodiments, the method of genetically modifying a TIL population comprises the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in the following: levine et al, 2006,103,17372-77, proc. Natl. Acad. Sci. USA; zufferey et al, nature Biotechnology 1997,15,871-75; dull et al, journal of virology 1998,72,8463-71 and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises the step of γ -retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, current protocols in molecular biology, 1996,9.9.1-9.9.16, the disclosures of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises the step of transposon mediated gene transfer. Transposon mediated gene transfer systems are known in the art, including those in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long term expression of the transposase does not occur in transgenic cells, such as transposase provided as mRNA (e.g., mRNA comprising a cap and a polyadenylation tail). Including salmon-like Tel-like transposases (SB or sleeping beauty transposases (e.g., SB10, SB11, and SB100 x)); and suitable transposon mediated gene transfer systems of enzymes with increased engineered enzyme activity are described, for example, in the following: hackett et al, molecular therapy 2010,18,674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

In some embodiments, transiently altering protein expression in TIL is induced by a small interfering RNA (SMALL INTERFERING RNA; siRNA), sometimes referred to as a short interfering RNA or silencing RNA, which is a double-stranded RNA molecule, typically 19-25 base pairs in length. siRNA is used in RNA interference (RNA INTERFERENCE; RNAi), wherein the siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence.

In some embodiments, transiently altering protein expression is a reduction in expression. In some embodiments, transiently altered protein expression is induced by self-delivering RNA interference (self-DELIVERING RNAINTERFERENCE; sdRNA) that is a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or-OCH ₃) that comprises an antisense (guide) strand of 20 nucleotides and a sense (sense) strand of 13 to 15 bases conjugated to cholesterol at its 3' end using a tetraethyl glycol (TEG) linker. Small interfering RNAs (sirnas), sometimes referred to as short interfering RNAs or silencing RNAs, are double-stranded RNA molecules, typically 19 to 25 base pairs in length. siRNA is used in RNA interference (RNAi), where the siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence. sdrnas are RNAi compounds that do not require covalent and hydrophobic modifications of the delivery vehicle to enter the cell. sdrnas are generally asymmetric chemically modified nucleic acid molecules with very small double stranded regions. The sdRNA molecules typically contain single-stranded and double-stranded regions, and may contain various chemical modifications within the single-stranded and double-stranded regions of the molecule. In addition, as described herein, the sdRNA molecules can be linked to hydrophobic conjugates, such as conventional and higher sterol molecules. sdrnas and related methods of making such sdrnas have also been widely described, for example, in the following: U.S. patent application publication nos. US2016/0304873 A1, US2019/0211337 A1, US 2009/013360 A1 and US2019/0048341A1, and US patent nos. 10,633,654 and 10,913,948B2, the respective disclosures of which are incorporated herein by reference. To optimize sdRNA structure, chemistry, targeting location, sequence preference, etc., an algorithm has been developed and used for sdRNA efficacy prediction. Based on these analyses, functional sdRNA sequences are generally defined as expression decreases by more than 70% at a concentration of 1 μm, with a probability of more than 40%.

Double-stranded RNA (dsRNA) may be generally used to define any molecule comprising a pair of complementary RNA strands, typically sense (sense) and antisense (guide) strands, and may include a single-stranded overhang (overhang) region. Unlike siRNA, the term dsRNA generally refers to a precursor molecule that includes the sequence of an siRNA molecule that is released from a larger dsRNA molecule by the action of a lyase system (including Dicer).

In some embodiments, the methods comprise transiently altering protein expression in a population of TILs, including TILs modified to express CCR, including the use of self-delivering RNA interference (sdRNA), which is a chemically synthesized asymmetric siRNA duplex, e.g., with a high percentage of 2'-OH substitutions (typically fluoro or-OCH 3), comprising an antisense (guide) strand of 20 nucleotides and a 13 to 15 base sense (follower) strand conjugated to cholesterol at its 3' end using a tetraethyl glycol (TEG) linker. Methods of using siRNA and sdRNA have been described in the following: khvorova and Watts, nature Biotechnology (Nat. Biotechnol.) 2017,35,238-248; byrne et al, J.Ocul.Pharmacol.Ther.), "2013,29,855-864; and Ligtenberg et al, molecular therapy 2018,26,1482-93, the disclosures of which are incorporated herein by reference. In some embodiments, the delivery of siRNA is accomplished using electroporation or cell membrane disruption (e.g., extrusion or SQZ method). In some embodiments, delivery of the sdrnas to the TIL population is not accomplished using electroporation, SQZ, or other methods, in effect the TIL population is exposed to sdrnas in medium at a concentration of 1 μm/10,000 TIL using a period of 1 to 3 days. In certain embodiments, the method comprises delivering siRNA or sdRNA to a population of TILs comprising exposing the population of TILs to sdRNA in a culture medium at a concentration of 1 μm/10,000 TILs for a period of time between 1 to 3 days. In some embodiments, delivering the sdRNA to the TIL population is accomplished using a period of 1 to 3 days to expose the TIL population to sdRNA in a medium at a concentration of 10 μm/10,000 TILs. In some embodiments, delivering the sdRNA to the TIL population is accomplished using a period of 1 to 3 days to expose the TIL population to sdRNA in a medium at a concentration of 50 μm/10,000 TILs. In some embodiments, delivering the sdRNA to the TIL population is accomplished using a period of 1 to 3 days to expose the TIL population to the sdRNA in the medium at a concentration of between 0.1 μm/10,000 TIL and 50 μm/10,000 TIL. In some embodiments, delivering the sdRNA to the TIL population is accomplished using a period of 1 to 3 days to expose the TIL population to the sdRNA in the medium at a concentration of between 0.1 μm/10,000 TIL and 50 μm/10,000 TIL, wherein the exposing to the sdRNA is performed two, three, four, or five times by adding fresh sdRNA to the medium. Other suitable processes are described, for example, in the following: U.S. patent application publication Nos. US2011/0039914 A1, US 2013/01331141 A1 and US 2013/013142 A1, and U.S. patent 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, the siRNA or sdRNA is inserted into the TIL population during manufacture. In some embodiments, the sdRNA encodes RNA that interferes with: NOTCH 1/2ICD, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGF beta, TGFBR2, cAMP Protein Kinase A (PKA), BAFF BR3, CISH and/or CBLB. In some embodiments, the reduction in expression is determined based on the percentage of gene silencing assessed, for example, by flow cytometry and/or qPCR. In some embodiments, expression is reduced by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

Self-deliverable RNAi technology based on chemically modified siRNA can be used in the methods of the invention to successfully deliver sdRNAs to TIL as described herein. The combination of backbone modifications with asymmetric siRNA structures and hydrophobic ligands (see, e.g., ligtenberg et al, molecular therapy 2018,26,1482-93 and U.S. patent application publication 2016/0304873A1, the disclosures of which are incorporated herein by reference) allows for the permeation of cultured mammalian cells by simple addition of sdrnas to a culture medium, utilizing nuclease stability or sdrnas without additional formulations and methods. This stability allows for supporting constant levels of RNAi-mediated reduction of target gene activity simply by maintaining an effective concentration of sdRNA in the medium. While not being bound by theory, backbone stabilization of sdrnas provides an extended reduced gene expression effect that can last for months in non-dividing cells.

In some embodiments, a more than 95% TIL transfection efficiency and reduced expression of the target is performed by each specific siRNA or sdRNA. In some embodiments, siRNA or sdRNA containing several unmodified ribose residues is replaced with a fully modified sequence to increase the efficacy and/or longevity of the RNAi effect. In some embodiments, the expression reduction effect is maintained for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the expression reduction effect decreases after 10 days or more of treatment of TIL with siRNA or sdRNA. In some embodiments, target expression is maintained with a reduction in expression of more than 70%. In some embodiments, target expression in the TIL is maintained with a reduction in expression of more than 70%. In some embodiments, reduced expression in the PD-1/PD-L1 pathway allows the TIL to exhibit a more potent in vivo effect, in some embodiments because of avoiding the inhibitory effect of the PD-1/PD-L1 pathway. In some embodiments, the decrease in PD-1 expression of siRNA or sdRNA results in increased proliferation of TIL.

In some embodiments, the sdRNA sequences used in the invention exhibit a 70% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the invention exhibit 75% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the invention exhibit 80% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in target gene expression of 85%. In some embodiments, the sdRNA sequences used in the invention exhibit a 90% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the invention exhibit a 95% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the invention exhibit 99% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of a target gene when delivered at a concentration of about 0.25 μm to about 4 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 0.25 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of a target gene when delivered at a concentration of about 0.5 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 0.75 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of a target gene when delivered at a concentration of about 1.0 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of a target gene when delivered at a concentration of about 1.25 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of a target gene when delivered at a concentration of about 1.5 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of a target gene when delivered at a concentration of about 1.75 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.0 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.25 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.5 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.75 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.0 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.25 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.5 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.75 μm. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced expression of the target gene when delivered at a concentration of about 4.0 μm.

In some embodiments, the siRNA or sdRNA oligonucleotide agent comprises one or more modifications to increase the stability and/or effectiveness of the therapeutic agent and to achieve efficient delivery of the oligonucleotide to the cell or tissue to be treated. Such modifications may include 2' -O-methyl modifications, 2' -O-fluoro modifications, dithiophosphate modifications, 2' F modified nucleotides, 2' -O-methyl modified and/or 2' deoxynucleotides. In some embodiments, the oligonucleotides are modified to include more than one hydrophobic modification, including, for example, sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and/or phenyl. In some embodiments, the chemically modified nucleotide is a combination of phosphorothioate, 2 '-O-methyl, 2' deoxy, hydrophobic modification, and phosphorothioate. In some embodiments, the sugar may be modified and the modified sugar may include, but is not limited to, D-ribose, 2' -O-alkyl (including 2' -O-methyl and 2' -0-ethyl), i.e., 2' -alkoxy, 2' -amino, 2' -S-alkyl, 2' -halo (including 2' -fluoro), T-methoxyethoxy, 2' -allyloxy (-OCH ₂CH＝CH₂), 2' -propargyl, 2' -propyl, ethynyl, ethenyl, propenyl, and cyano, and the like. In some embodiments, the sugar moiety may be a hexose and be incorporated into an oligonucleotide, such as Augustyns et al, nucleic acids research (nucleic acids res.) 1992,18,4711, the disclosure of which is incorporated herein by reference.

In some embodiments, the double stranded siRNA or sdRNA oligonucleotides of the invention are double stranded throughout their length, i.e., have no protruding single stranded sequence at either end of the molecule, i.e., are blunt ended. In some embodiments, individual nucleic acid molecules may have different lengths. In other words, the double stranded siRNA or sdRNA oligonucleotides of the invention are not double stranded throughout their length. For example, when two separate nucleic acid molecules are used, one of the molecules, e.g., a first molecule comprising an antisense sequence, may be longer than a second molecule to which it hybridizes (leaving a portion of the molecule single stranded). In some embodiments, when single nucleic acid molecules are used, a portion of the molecules at either end may remain single stranded.

In some embodiments, the double stranded siRNA or sdRNA oligonucleotides of the invention contain mismatches and/or loops or bulges but are double stranded over at least about 70% of the length of the oligonucleotide. In some embodiments, the double-stranded oligonucleotides of the invention are double-stranded over at least about 80% of the length of the oligonucleotide. In other embodiments, the double stranded siRNA or sdRNA oligonucleotides of the invention are double stranded over at least about 90% -95% of the length of the oligonucleotide. In some embodiments, the double stranded siRNA or sdRNA oligonucleotides of the invention are double stranded over at least about 96% -98% of the length of the oligonucleotide. In some embodiments, the double stranded oligonucleotides of the invention contain at least or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

In some embodiments, the siRNA or sdRNA oligonucleotide may be substantially protected from nucleases, e.g., by modifying 3 'or 5' linkages, as described in U.S. patent No. 5,849,902 and WO 98/13526, the disclosures of which are incorporated herein by reference. For example, an oligonucleotide may be resistant by inclusion of a "blocking group". The term "blocking group" as used herein refers to a substituent (e.g., other than an OH group) that can be attached to an oligonucleotide or core monomer as a protecting or coupling group for synthesis (e.g., FITC, propyl (CH ₂-CH₂-CH₃), diol (-O-CH ₂-CH₂ -O-) phosphate (PO ₃ ^2"), hydrogen phosphate, or amino phosphite). "blocking groups" may also include "terminal blocking groups" or "exonuclease blocking groups" that protect the 5 'and 3' ends of oligonucleotides, including modified nucleotide and non-nucleotide exonuclease resistant structures.

In some embodiments, at least a portion of the contiguous polynucleotides within the siRNA or sdRNA are linked by a substituent linkage, such as a phosphorothioate linkage.

In some embodiments, the chemical modification may result in an increase in uptake of the siRNA or sdRNA by at least 1.5、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、225、250、275、300、325、350、375、400、425、450、475 or 500% of the cells. In some embodiments, at least one of the C or U residues comprises a hydrophobic modification. In some embodiments, the plurality of C and U contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U may contain hydrophobic modifications. In some embodiments, all C and U contain hydrophobic modifications.

In some embodiments, the siRNA or sdRNA molecule exhibits enhanced endosomal release by incorporation of a protonatable amine. In some embodiments, the protonatable amine is incorporated into the sense strand (in the portion of the molecule that is discarded after RISC loading). In some embodiments, the siRNA or sdRNA compounds of the invention comprise an asymmetric compound comprising a duplex region (10 to 15 bases long required for efficient RISC entry) and a single stranded region of 4-12 nucleotides long; having a 13 nucleotide duplex. In some embodiments, a single stranded region of 6 nucleotides is employed. In some embodiments, the single stranded region of the siRNA or sdRNA comprises 2 to 12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). In some embodiments, 6 to 8 phosphorothioate internucleotide linkages are employed. In some embodiments, the siRNA or sdRNA compounds of the invention also include unique chemical modification patterns that provide stability and are compatible with RISC entry. For example, the guide strand may also be modified by any chemical modification that demonstrates stability without interfering with RISC entry. In some embodiments, the pattern of chemical modification into the guide strand includes C and U nucleotides that are mostly 2'f modified and 5' phosphorylated.

In some embodiments, at least 30% of the nucleotides in the siRNA or sdRNA are modified. In some embodiments, at least 30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% of the nucleotides in the siRNA or sdRNA are modified. In some embodiments, 100% of the nucleotides in the siRNA or sdRNA are modified.

In some embodiments, the siRNA or sdRNA molecule has few double stranded regions. In some embodiments, the double-stranded region of the molecule is in the range of 8 to 15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9,10, 11, 12, 13, 14, or 15 nucleotides long. In some embodiments, the duplex region is 13 nucleotides long. There may be 100% complementarity between the guide strand and the follower strand, or there may be more than one mismatch between the guide strand and the follower strand. In some embodiments, on one end of the double-stranded molecule, the molecule is blunt-ended or has an overhang of one nucleotide. The single-stranded region of the molecule is in some embodiments between 4 and 12 nucleotides long. In some embodiments, the single stranded region may be 4, 5,6,7,8, 9,10, 11, or 12 nucleotides long. In some embodiments, the single stranded region may also be less than 4 nucleotides or greater than 12 nucleotides long. In certain embodiments, the single stranded region is 6 or 7 nucleotides long.

In some embodiments, the siRNA or sdRNA molecule has increased stability. In some cases, the chemically modified siRNA or sdRNA molecule has a half-life in culture medium longer than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours, including any intermediate value. In some embodiments, the half-life of the siRNA or sd-RNA in the medium is more than 12 hours.

In some embodiments, the siRNA or sdRNA is optimized to increase potency and/or reduce toxicity. In some embodiments, the nucleotide length of the guide and/or the satellite strand and/or the number of phosphorothioate modifications in the guide and/or the satellite strand may affect the potency of the RNA molecule in some aspects, while substitution of 2 '-fluoro (2' f) modification with a2 '-O-methyl (2' ome) modification may affect the toxicity of the molecule in some aspects. In some embodiments, it is expected that reducing the 2' f content of a molecule will reduce the toxicity of the molecule. In some embodiments, the number of phosphorothioate modifications in the RNA molecule can affect the efficiency of uptake of the molecule into a cell, e.g., passive uptake of the molecule into a cell. In some embodiments, the siRNA or sdRNA does not have a 2' f modification, but is characterized by equal efficacy in terms of cellular uptake and tissue penetration.

In some embodiments, the guide strand is about 18 to 19 nucleotides in length and has about 2 to 14 phosphate modifications. For example, the guide strand may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 nucleotides modified with phosphate. The guide chain may contain more than one modification that imparts increased stability without interfering with RISC entry. Phosphate modified nucleotides, such as phosphorothioate modified nucleotides, may be at the 3 'end, the 5' end, or distributed throughout the guide strand. In some embodiments, 10 nucleotides to the 3' end of the guide strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate modified nucleotides. The guide strand may also contain 2'F and/or 2' OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at position one in the guide strand (the nucleotide in the 5 'most position to the guide strand) is modified and/or phosphorylated by 2' ome. The C and U nucleotides in the guide strand may be modified by 2' F. For example, the C and U nucleotides in positions 2 to 10 of the 19 nucleotide guide strand (or corresponding positions in the guide strand of different lengths) may be modified with a 2' F. The C and U nucleotides in the guide strand may also be modified with 2' OMe. For example, the C and U nucleotides in positions 11 to 18 of the l9 nucleotide guide strand (or corresponding positions in the different length guide strand) may be modified by a 2' OMe. In some embodiments, the nucleotide at the 3' -most end of the guide strand is unmodified. In certain embodiments, a majority of C and U within the guide strand is modified by 2'F and phosphorylated to the 5' end of the guide strand. In other embodiments, the C or U in positions 1 and 11-18 is modified by a 2'OMe and phosphorylated to the 5' end of the guide strand. In other embodiments, the C or U in positions 1 and 11-18 is modified with a 2' OMe, phosphorylated to the 5' end of the guide strand, and the C or U in positions 2-10 is modified with a 2' F.

Self-deliverable RNAi technology provides a method of directly transfecting cells with an RNAi agent (whether siRNA, sdRNA, or other RNAi agent) without the need for additional agents or technologies. The ability to transfect difficult to transfect cell lines, high in vivo activity and ease of use are features of the compositions and methods that present significant functional advantages over traditional siRNA-based techniques, and thus employ the sdRNA method in several embodiments of methods for reducing target gene expression in the TILs of the present invention. The sdRNA method allows for the direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues in vitro and in vivo. The sdrnas described in some embodiments of the invention herein are available from ADVIRNA LLC of the united states, mass, wood.

SiRNA and sdRNA can be formed in hydrophobically modified siRNA-antisense oligonucleotide hybridization structures, as disclosed, for example, in Byrne et al, journal of ophthalmic pharmacological treatment (j. OcularPharmacol. Therapeutic.), 2013,29,855-864, the disclosure of which is incorporated herein by reference.

In some embodiments, siRNA or sdRNA oligonucleotides can be delivered to the TILs described herein using sterile electroporation. In certain embodiments, the method comprises aseptically electroporating the TIL population to deliver the siRNA or sdRNA oligonucleotides.

In some embodiments, the oligonucleotide may be delivered to the cell in combination with a transmembrane delivery system. In some embodiments, the transmembrane delivery system comprises a lipid, a viral vector, and the like. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent without any delivery agent. In certain embodiments, the method comprises using a transmembrane delivery system to deliver the siRNA or sdRNA oligonucleotide to the TIL population.

The oligonucleotides and oligonucleotide compositions are contacted with (e.g., contacted with, also referred to herein as administered or delivered to) the TILs described herein and ingested, including passively ingested by the TILs. sdRNA may be added to TIL as described herein when: during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D), after step D and before collection in step E, during or after collection in step F, before or during final formulation and/or transfer to an infusion bag in step F, and before any optional cryopreservation step in step F. In addition, siRNA or sdRNA may be added after thawing from any of the cryopreservation steps in step F. In some embodiments, one or more sdrnas targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, TET2, and CBLB) may be added to a cell culture medium comprising TIL and other reagents at a concentration selected from 100nM to 20mM, 200nM to 10mM, 500nM to 1mM, 1 μΜ to 100 μΜ, and 1 μΜ to 100 μΜ. In some embodiments, one or more sdrnas targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, TET2, and CBLB) may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: 0.1. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, 0.5. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, 0.75. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, 1. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, 1.25. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, 1.5. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, 2. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, 5. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium, or 10. Mu.M siRNA or sdRNA per 10,000 TIL/100. Mu.L medium. In some embodiments, more than one sdRNA targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, TET2, and CBLB) may be added to the TIL culture twice a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the pre-REP or REP phase.

The oligonucleotide compositions of the invention, including the sdrnas, may be contacted with TIL as described herein during the amplification process, for example, by dissolving high concentrations of the sdrnas in cell culture medium and allowing sufficient time for passive uptake to occur. In certain embodiments, the methods of the invention comprise contacting a population of TILs with an oligonucleotide composition as described herein. In certain embodiments, the method comprises dissolving an oligonucleotide, e.g., a sdRNA, in a cell culture medium and contacting the cell culture medium with a population of TILs. The TIL may be a first population, a second population, and/or a third population as described herein.

In some embodiments, delivery of the oligonucleotide into the cell may be enhanced by suitable art-recognized methods, including calcium phosphate, DMSO, glycerol, or polydextrose, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes, using methods known in the art, e.g., those described in: U.S. patent No. 4,897,355; no. 5,459,127; 5,631,237 th sheet; 5,955,365 th sheet; 5,976,567 th sheet; 10,087,464 th sheet; and 10,155,945; and Bergan et al, nucleic acids reviews (nucleic acids res.) 1993,21,3567, the disclosures of each of which are incorporated herein by reference.

In some embodiments, more than one siRNA or sdRNA is used to reduce target gene expression. In some embodiments, one or more of PD-1, TIM-3, CBLB, LAG3, CTLA-4, TIGIT, TET2 and/or CISH targeted to siRNA or sdRNA are used together. In some embodiments, PD-1siRNA or sdRNA is used with one or more of TIM-3, CBLB, LAG3, CTLA-4, TIGIT, TET2, and/or CISH to reduce expression of more than one gene target. In some embodiments, LAG3 siRNA or sdRNA is used in combination with CISH targeting the siRNA or sdRNA to reduce expression of both target genes. In some embodiments, one or more siRNAs or sdRNAs targeting PD-1, TIM-3, CBLB, LAG3, CTLA-4, TIGIT, TET2, and/or CISH herein are commercially available from ADVIRNA LLC of WORS, mass.

In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGF beta R2, PKA, CBLB, BAFF (BR 3) and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGF beta R2, PKA, CBLB, BAFF (BR 3) and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1 and the other siRNA or sdRNA targets a gene selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, tgfβr2, PKA, CBLB, BAFF (BR 3), and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of: PD-1, LAG-3, CISH, CBLB, TIM, CTLA-4, TIGIT, TET2, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from PD-1 and one of: LAG3, CISH, CBLB, TIM, and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets CBLB. For some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets CBLB. For some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets CBLB. For some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets PD-1. For some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets LAG3. For some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets CBLB. For some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets CTLA-4. For some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets CTLA-4 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets CTLA-4 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets TIGIT and one siRNA or sdRNA targets TET2.

As described above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been genetically modified by gene editing to enhance their therapeutic effects. Embodiments of the invention encompass gene editing by nucleotide insertion (RNA or DNA) into a TIL population to facilitate expression of and inhibit expression of one or more proteins, and combinations thereof also provide methods for amplifying TIL into a therapeutic population, wherein the methods comprise gene editing the TIL. There are several gene editing techniques available for genetically modifying a population of TILs, which are suitable for use in accordance with the present invention. Such methods include the methods described below and viral and transposon methods described elsewhere herein. In some embodiments, the method of genetically modifying a TIL, MILs, or PBL to express CCR may also include modifications that inhibit gene expression by stabilizing gene knockouts or temporary gene attenuation of such genes.

In some embodiments, the method comprises a method of genetically modifying a population of TILs, the population of TILs being a first population, a second population, and/or a third population as described herein. In some embodiments, the method of genetically modifying a population of TILs comprises the step of stably incorporating genes for producing or inhibiting (e.g., silencing) more than one protein. In some embodiments, the method of genetically modifying a population of TILs comprises the step of electroporation. Electroporation methods are known in the art and are described, for example, in the following: tsong J biophysics 1991,60,297-306 and U.S. patent application publication No. 2014/0227237A1, the respective disclosures of which are incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in the following: U.S. Pat. nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or transient changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or transient changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or transient changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or transient changes in the TIL, comprising the step of applying a series of at least three single, operator controlled independently programmed DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein a first pulse interval of two of the first set of at least three pulses is different from a second pulse interval of two of the second set of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, comprising the step of applying a series of at least three DC electric pulses (field strength equal to or greater than 100V/cm) to the TIL, wherein the series of at least three DC electric pulses has one, two, or three of the following characteristics: (1) At least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval of two of the first set of the at least three pulses is different from a second pulse interval of two of the second set of the at least three pulses such that the induced pores last for a relatively long period of time and maintain survival of the TIL. In some embodiments, the method of genetically modifying a TIL population comprises the step of calcium phosphate transfection. The calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in the following: graham and van der Eb, virology 1973,52,456-467; wigler et al, 1979,76,1373-1376 in the national academy of sciences of the United states; and Chen and Okayarea, molecular cell biology 1987,7,2745-2752; and U.S. patent No. 5,593,875, the disclosures of each of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs comprises a step of lipofection. Methods of liposome transfection, such as 1:1 (w/w) liposome formulations employing the cationic lipids N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) in filtered water are known in the art and are described in Rose et al, biotechnology 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA, 1987,84,7413-7417, and U.S. Pat. Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484 and 7,687,070, the respective disclosures of which are each incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises the step of transfection using the method described in: U.S. patent nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of each of which are incorporated herein by reference. The TIL may be a first TIL population, a second TIL population, and/or a third TIL population as described herein.

According to an embodiment, the gene editing method may comprise the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at more than one immune checkpoint gene. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on recognition of specific DNA sequences within the genome to target the nuclease domain to this location and mediate the creation of double-strand breaks at the target sequence. Double strand breaks in DNA then recruit endogenous repair systems to the break site to mediate genome editing through non-homologous end joining (NHEJ) or Homology Directed Repair (HDR). Thus, repair of the break may result in the introduction of insertion/deletion mutations that disrupt (e.g., silence, inhibit or enhance) the target gene product.

The main classes of nucleases developed to enable site-specific genome editing include Zinc Finger Nucleases (ZFNs), transcription activator-like nucleases (TALENs) and CRISPR-associated nucleases (e.g., CRISPR/Cas 9). These nuclease systems can be broadly classified into two classes based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, whereas CRISPR systems, such as Cas9, target specific DNA sequences through short RNA guide molecules that base pair directly with target DNA and through protein-DNA interactions. See, for example, cox et al, nature Medicine 2015, volume 21, phase 2.

Non-limiting examples of gene editing methods that may be used in accordance with the TIL amplification methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, which are described in more detail below. According to one embodiment, the method of amplifying a TIL into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., gen 2 process) or as described in U.S. patent application publication nos. US2020/0299644 A1 and US 2020/011719 A1 and US patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein the method further comprises genetically editing at least a portion of the TIL by one or more of the CRISPR method, TALE method, or ZFN method to produce a TIL that may provide enhanced therapeutic effects. According to an embodiment, the improved therapeutic effect of the gene-edited TIL may be assessed by comparing the gene-edited TIL with unmodified TIL in vitro, e.g., by assessing in vitro effector function, cytokine profile, etc., as compared to unmodified TIL. In certain embodiments, the method comprises gene editing the TIL population using CRISPR, TALE, and/or ZFN methods.

The method for amplifying a TIL into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., gen 2) or as described in U.S. patent application publication nos. US2020/0299644 A1 and US 2020/011719 A1 and US patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein the method further comprises editing at least a portion of the TIL by a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf 1) gene. According to particular embodiments, the use of a CRISPR method during a TIL amplification process results in silencing or reducing expression of one or more immune checkpoint genes in at least a portion of a therapeutic TIL population. Alternatively, the use of a CRISPR method during a TIL amplification process results in enhanced expression of one or more immune checkpoint genes in at least a portion of a therapeutic TIL population.

CRISPR stands for clustered regularly interspaced short palindromic repeats. The method of gene editing using a CRISPR system is also referred to herein as the CRISPR method. There are three types of CRISPR systems that incorporate RNA and Cas proteins and can be used according to the present invention: I. type II and III. Type II CRISPR (exemplified by Cas 9) is one of the most well characterized systems.

CRISPR technology is an adaptation of the natural defense mechanisms from bacteria and archaea (domains of unicellular microorganisms). These organisms use CRISPR-derived RNAs and various Cas proteins (including Cas 9) to prevent attack by viruses and other exosomes by chopping and disrupting the DNA of the foreign intruder. CRISPR is a DNA-specific region with two unique features: nucleotide repeats and spacers are present. The repeated sequence of nucleotides is distributed throughout the CRISPR region with short foreign DNA segments (spacers) interspersed in the repeated sequence. In a type II CRISPR/Cas system, the spacer is integrated within the CRISPR genomic locus and transcribed and processed into short CRISPR RNA (crRNA). These crrnas anneal to transactivation crRNA (tracrRNA) and guide Cas proteins for sequence-specific cleavage and silencing of pathogenic DNA. Target recognition by Cas9 proteins requires a "seed" sequence within the crRNA and a conserved Protospacer Adjacent Motif (PAM) sequence containing dinucleotides upstream of the crRNA binding region. Thus the CRISPR/Cas system can be re-targeted by redesigning the crRNA to cleave almost any DNA sequence. CrRNA and tracrRNA in the primordial system can be reduced to about 100 nucleotide single guide RNAs (sgrnas) for genetic engineering. The CRISPR/Cas system can be carried directly into human cells by co-delivering plasmids expressing Cas9 endonuclease and essential crRNA components. Different Cas protein variants may be used to reduce targeting limitations (e.g., heterologous homologs of Cas9, such as Cpf 1).

Non-limiting examples of genes that can be enhanced by permanent gene editing of TIL by CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18 and IL-21.

Examples of systems, methods, and compositions for altering expression of a target gene sequence by a CRISPR method and that can be used in accordance with embodiments of the present invention are described in U.S. patent nos. 8,697,359, 8,993,233, 8,795,965, 8,771,945, 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 8,871,445, 8,906,616, and 8,895,308, the disclosures of each of which are incorporated herein by reference. Resources for performing the CRISPR method, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are available from companies such as gold srey (GenScript).

In some embodiments, genetically modifying a population of TILs as described herein may be performed using a CRISPR/Cpf1 system as described in U.S. patent No. 9790490, the disclosure of which is incorporated herein by reference.

The method for amplifying a TIL into a therapeutic population may be performed according to any of the embodiments of the methods described herein (e.g., gen 2) or as described in U.S. patent application publication nos. US2020/0299644 A1 and US 2020/011719 A1 and US patent No.10,925,900 (the disclosures of which are incorporated herein by reference), wherein the method further comprises gene editing at least a portion of the TIL by the TALE method. According to particular embodiments, use of the TALE method during the TIL amplification process results in silencing or reducing expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, use of the TALE method during the TIL amplification process results in enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALEs represent transcription activator-like effector proteins, which include transcription activator-like effector nucleases (TALENs). The method of gene editing using the TALE system is also referred to herein as the TALE method. TALE is a naturally occurring protein from the plant pathogenic bacterium xanthomonas that contains a DNA binding domain consisting of a series of repeat domains of 33 to 35 amino acids each recognizing a single base pair. TALE specificity is determined by two hypervariable amino acids called Repeated Variable Diradicals (RVDs). Modular TALE repeats are linked together to recognize a contiguous DNA sequence. Specific RVDs in the DNA binding domain recognize bases in the target locus, providing structural features to assemble a predictable DNA binding domain. The DNA binding domain of TALE is fused to the catalytic domain of a fokl endonuclease type IIS to prepare a targetable TALE nuclease. To induce site-specific mutations, two separate TALEN arms separated by a 14 to 20 base pair spacer region draw the fokl monomer closer to dimerize and create a targeted double strand break.

Several large systematic studies using various assembly methods indicate that TALE repeat sequences can be incorporated to identify virtually any user-defined sequence. Custom designed TALE arrays are also commercially available from CELLECTIS BIORESEARCH (paris, france), transposagen Biopharmaceuticals (lekurd, kentucky, usa) and Life Technologies (gland island, new york, usa). TALE and TALEN processes suitable for use in the present invention are described in U.S. patent application publication nos. US 2011/0201118A1, US 2013/0177869 A1, US2013/0315884 A1, US 2015/0203871A1 and US 2016/012596 A1, the respective disclosures of which are incorporated herein by reference.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TIL by TALE methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering expression of a target gene sequence by the TALE method are described in U.S. patent No. 8,586,526, which is incorporated herein by reference, which can be used in accordance with embodiments of the present invention.

The method for amplifying a TIL into a therapeutic population may be performed according to any of the embodiments of the methods described herein or as described in U.S. patent application publication nos. US2020/0299644 A1 and US 2020/011719 A1 and U.S. patent No. 10,925,900 (the disclosures of which are incorporated herein by reference), wherein the method further comprises genetically editing at least a portion of the TIL by a zinc finger or zinc finger nuclease method. According to particular embodiments, the use of zinc finger methods during the TIL amplification process results in silencing or reducing expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of zinc finger methods during the TIL amplification process results in enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

The individual zinc fingers contain about 30 amino acids in a conserved ββα configuration. Several amino acids on the alpha-helical surface typically contact 3bp in the major groove of DNA at different levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA binding domain that includes eukaryotic transcription factors and contains zinc fingers. The second domain is a nuclease domain that includes fokl restriction enzymes and is responsible for catalytic cleavage of DNA.

The DNA binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and each can recognize between 9 and 18 base pairs. If the zinc finger domain is specific for its intended target site, even a pair of 3-finger ZFNs recognizing a total of 18 base pairs can theoretically target a single locus in the mammalian genome. One approach to creating new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The most common module assembly process involves combining three separate zinc fingers, each recognizing a3 base pair DNA sequence, to produce a3 finger array that recognizes 9 base pair target sites. Alternatively, a selection-based approach (e.g., oligo library engineering) can be used to select new zinc finger arrays from random libraries that take into account background-dependent interactions between adjacent fingers. Engineered zinc fingers are commercially available from Sangamo Biosciences (Richmong, california) and Sigma-Aldrich (St. Louis, misu, USA).

Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TIL by zinc finger methods include PD-1、CTLA-4、LAG-3、HAVCR2(TIM-3)、Cish、TGFβ、PKA、CBL-B、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、BTLA、CD160、TIGIT、TET2、CD96、CRTAM、LAIR1、SIGLEC7、SIGLEC9、CD244、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT1、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2、GUCY1B3、TOX、SOCS1、ANKRD11 and BCOR.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TIL by zinc finger methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-18, and IL-21.

Examples of systems, methods, and compositions for altering expression of a target gene sequence by a zinc finger method and that can be used in accordance with embodiments of the present invention are described in U.S. Pat. nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, each of which is incorporated herein by reference.

Examples of systems, methods, and compositions for altering expression of target gene sequences by zinc finger methods and which may be used in accordance with other embodiments of the present invention are described in Beane et al, molecular therapy, 2015,23,1380-1390, the disclosure of which is incorporated herein by reference.

In some embodiments, the TIL is optionally genetically engineered to include other functionalities including, but not limited to, a high affinity TCR, such as a TCR that targets a tumor associated antigen (e.g., MAGE-1, HER2, or NY-ESO-1), or a Chimeric Antigen Receptor (CAR) that binds to a tumor associated cell surface molecule (e.g., mesothelin) or a lineage-restricted cell surface molecule (e.g., CD 19). In certain embodiments, the methods comprise genetically engineering a population of TILs to comprise a high affinity TCR, e.g., a TCR that targets a tumor-associated antigen (e.g., MAGE-1, HER2, or NY-ESO-1), or a Chimeric Antigen Receptor (CAR) that binds to a tumor-associated cell surface molecule (e.g., mesothelin) or a lineage-restricted cell surface molecule (e.g., CD 19). Suitably, the TIL population may be a first population, a second population, and/or a third population as described herein.

D. closed system for TIL manufacture

The present invention provides for the use of a closed system during the TIL cultivation process. Such a closed system allows for prevention and/or reduction of microbial contamination, allows for fewer culture flasks to be used and allows for cost reduction. In some embodiments, the closure system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http:// www.fda.gov/cber/guides.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

The aseptic connection device (STCD) creates an aseptic weld between two compatible pieces of tubing. This procedure allows sterile connection of multiple containers and tube diameters. In some embodiments, the containment system includes a luer lock (luer lock) and heat seal system as described in the examples. In some embodiments, the containment system is accessed through a syringe under sterile conditions to maintain sterility and containment properties of the system. In some embodiments, a closed system as described in the examples is employed. In some embodiments, the TIL is formulated into a final product formulation vessel according to the methods described in the examples herein.

In some embodiments, the closed system uses one container from the time the tumor fragment is obtained to the time it is ready to administer TIL to the patient or to be cryopreserved. In some embodiments, when two containers are used, the first container is a closed G container, and the TIL population is centrifuged and transferred to the infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is one containing HypoThermosol. The closed system or closed TIL cell culture system is characterized in that once a tumor sample and/or tumor debris has been added, the system is sealed tightly from the outside to form a closed environment, which is not contaminated with bacteria, fungi and/or any other microorganisms.

In some embodiments, the microbial contamination reduction is between about 5% and about 100%. In some embodiments, the microbial contamination reduction is between about 5% and about 95%. In some embodiments, the microbial contamination reduction is between about 5% and about 90%. In some embodiments, the microbial contamination reduction is between about 10% and about 90%. In some embodiments, the microbial contamination reduction is between about 15% and about 85%. In some embodiments, the microbial contamination reduction is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

The closed system allows the TIL to grow in the absence of microbial contamination and/or with significantly reduced microbial contamination.

In addition, the pH, partial pressure of carbon dioxide, and partial pressure of oxygen of the TIL cell culture environment each vary with cell culture. Therefore, even though a medium suitable for cell culture is circulated, a closed environment is continuously required to be maintained as an optimal environment for TIL proliferation. For this purpose, it is desirable to monitor physical factors of pH, partial pressure of carbon dioxide and partial pressure of oxygen in the culture solution of the closed environment by means of sensors, signals of which are used to control a gas exchanger installed at an inlet of the culture environment, and adjust the partial pressure of gas of the closed environment in real time according to changes in the culture solution so as to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system that incorporates a gas exchanger equipped with a monitoring device that measures the pH, partial pressure of carbon dioxide, and partial pressure of oxygen of the closed environment at the inlet to the closed environment, and optimizes the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring device.

In some embodiments, the pressure within the enclosed environment is controlled continuously or intermittently. That is, the pressure in the closed environment may be varied by means of, for example, a pressure maintenance device, thereby ensuring that the space is suitable for TIL growth under positive pressure conditions or promoting exudation of fluid under negative pressure conditions and thus promoting cell proliferation. Further, by intermittently applying the negative pressure, it is possible to uniformly and effectively displace the circulating liquid in the closed environment by temporarily reducing the volume of the closed environment.

In some embodiments, optimal culture components for TIL proliferation may be replaced or added, factors including, for example, IL-2 and/or OKT3, and combinations thereof may be added.

E. optional cryopreservation of TIL

The subject TIL population (e.g., the second TIL population) or the amplified TIL population (e.g., the third TIL population) may optionally be cryopreserved. In some embodiments, cryopreservation occurs in a therapeutic TIL population. In some embodiments, cryopreservation occurs at TIL collected after the second amplification. In some embodiments, cryopreservation occurs at TIL in exemplary step F of fig. 8 (e.g., fig. 8A and/or 8B and/or 8C and/or 8D, in particular). In some embodiments, the TIL is stored frozen in an infusion bag. In some embodiments, the TIL is cryopreserved prior to placement in the infusion bag. In some embodiments, the TIL is cryopreserved and not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is typically accomplished by placing the TIL population in a chilled solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in solution were placed in a cryovial and stored at-80 ℃ for 24 hours, where they were optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al, oncology report (Acta Oncologica) 2013,52,978-986.

Where appropriate, cells were removed from the freezer and thawed in a 37℃water bath until approximately 4/5 of the solution was thawed. The cells are typically resuspended in complete medium and optionally washed more than once. In some embodiments, thawed TILs may be calculated and survival assessed as known in the art.

In some embodiments, the TIL population is cryopreserved using CS10 cryopreservation media (CryoStor 10,BioLife Solutions). In some embodiments, the TIL population is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In some embodiments, TIL populations are cryopreserved using a CS10 to cell culture medium ratio of 1:1 (vol: vol). In some embodiments, the TIL population is cryopreserved using a CS10 to cell culture medium (further comprising additional IL-2) ratio of about 1:1 (vol: vol).

As described above and as illustrated in steps a to E provided in fig. 1 and/or 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D), cryopreservation may be performed at multiple points during TIL amplification. In some embodiments, the amplified TIL population after a first amplification (as provided, for example, according to step B) or after more than one second amplification according to step D of fig. 1 or 8 (particularly, for example, fig. 8A and/or 8B and/or 8C and/or 8D) may be cryopreserved. Cryopreservation can generally be accomplished by placing the TIL population in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% Dimethylsulfoxide (DMSO)). The cells in solution were placed in a cryovial and stored at-80 ℃ for 24 hours, where they were optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al, oncology report (Acta Oncologica) 2013,52,978-986. In some embodiments, TIL is stored frozen in 5% DMSO. In some embodiments, TIL is stored frozen in cell culture medium plus 5% DMSO. In some embodiments, the TIL is cryopreserved according to the methods provided in example 6.

In some cases, the TIL population of step B of fig. 1 or 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) may be immediately cryopreserved using the protocol discussed below. Or the subject TIL population may undergo steps C and D from fig. 1 or 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) and then be cryopreserved after step D of fig. 1 or 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). Similarly, in cases where a genetically modified TIL is to be used in therapy, the population of TILs from step B or step D of fig. 1 or 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D) may be genetically modified for appropriate treatment.

F. Phenotypic characterization of amplified TIL

In some embodiments, TIL is analyzed for expression of a variety of phenotypic markers after amplification, including those described herein and in the examples. In some embodiments, the expression of more than one phenotypic marker is examined. In some embodiments, the phenotypic characteristics of the TIL are analyzed after the first amplification in step B from fig. 1 or fig. 8 (in particular, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, the phenotypic characteristics of the TIL are analyzed during the transition in step C from fig. 1 or 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the phenotypic characteristics of the TIL are analyzed during the transition in step C from fig. 1 or fig. 8 (in particular, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D) and after cryopreservation. In some embodiments, the phenotypic characteristics of the TIL are analyzed after the second amplification in step D from fig. 1 or 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the phenotypic characteristics of the TIL are analyzed after two or more amplifications in step D from fig. 1 or fig. 8 (in particular, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D).

In some embodiments, the marker is selected from CD8 and CD28. In some embodiments, the expression of CD8 is examined. In some embodiments, the expression of CD28 is examined. In some embodiments, the expression of CD8 and/or CD28 on TIL produced according to the inventive process is higher compared to other processes (e.g., gen 3 processes as provided, for example, in fig. 8 (particularly, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D)) compared to 2A processes as provided, for example, in fig. 8 (particularly, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, the expression of CD8 on the TIL produced by the process according to the invention is higher compared to other processes (e.g., gen 3 process as provided, for example, in fig. 8 (particularly, e.g., fig. 8B)) compared to 2A process as provided, for example, in fig. 8 (particularly, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, the expression of CD28 on the TIL produced according to the present process is higher compared to other processes (e.g., gen 3 processes as provided, for example, in fig. 8 (particularly, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D)) compared to 2A processes as provided, for example, in fig. 8 (particularly, e.g., fig. 8A). In some embodiments, high CD28 expression indicates a younger, more durable TIL phenotype. In some embodiments, the expression of more than one regulatory marker is measured.

In some embodiments, during any step of the methods for expanding tumor-infiltrating lymphocytes (TILs) described herein, the first TIL population, the second TIL population, the third TIL population, or the collected TIL population is not selected based on CD8 and/or CD28 expression.

In some embodiments, the percentage of central memory cells of TIL produced by the process according to the invention is higher compared to other processes (e.g., gen 3 processes as provided, for example, in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), compared to 2A processes as provided, for example, in fig. 8 (particularly, e.g., fig. 8A). In some embodiments, the memory marker of the central memory cell is selected from CCR7 and CD62L.

In some embodiments, the cd4+ and/or cd8+ TIL memory subsets may be divided into different memory subsets. In some embodiments, the cd4+ and/or cd8+ TIL comprises an initial (cd45ra+cd62l+) TIL. In some embodiments, the CD4+ and/or CD8+ TILs comprise a central memory (CM; CD45 RA-CD62L+) TIL. In some embodiments, the CD4+ and/or CD8+ TILs comprise effector memory (EM; CD45RA-CD 62L-) TILs. In some embodiments, the CD4+ and/or CD8+ TILs include RA+ effect memory/effect (TEMRA/TEFF; CD45RA+CD62L+) TILs.

In some embodiments, TIL expresses more than one marker selected from the group consisting of: granzyme B, perforin and granulysin. In some embodiments, TIL expresses granzyme B. In some embodiments, the TIL expresses perforin. In some embodiments, TIL expresses granulysin.

In some embodiments, cytokine release from the restimulated TIL can also be assessed using a cytokine release assay. In some embodiments, the interferon-gamma (IFN-gamma) secretion of TIL may be assessed. In some embodiments, IFN-gamma secretion is measured by ELISA assays. In some embodiments, IFN- γ secretion is measured after a rapid second amplification step by ELISA assay, after step D as provided, for example, in fig. 8 (in particular, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL health is measured by IFN-gamma (IFN-gamma) secretion. In some embodiments, IFN-gamma secretion is indicative of active TIL. In some embodiments, potency assays for IFN-gamma production are employed. IFN-gamma production is another measure of cytotoxic potential. IFN-gamma production can be measured by determining the amount of cytokine IFN-gamma in TIL medium stimulated with antibodies against CD3, CD28 and CD137/4-1 BB. IFN-y levels in media from these stimulated TILs can be measured by assaying IFN-y release. In some embodiments, an increase in TIL of step D during Gen 3, e.g., as provided in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D), as compared to IFN- γ production of step D during 2A, e.g., as provided in fig. 8 (particularly, e.g., fig. 8A), indicates an increase in the cytotoxic potential of step D TIL. In some embodiments, IFN-gamma secretion is doubled, tripled, quadrupled or quintupling or more. In some embodiments, IFN-gamma secretion is doubled. In some embodiments, IFN-gamma secretion is increased by a factor of two. In some embodiments, IFN-gamma secretion is increased three times. In some embodiments, IFN-gamma secretion is increased four-fold. In some embodiments, IFN-gamma secretion is increased five-fold. In some embodiments, IFN-. Gamma.is measured using a Quantikine ELISA kit. In some embodiments, IFN-gamma is measured in ex vivo TIL. In some embodiments, IFN- γ is measured in ex vivo TILs, including TILs produced by methods of the invention (including, for example, the method of FIG. 8B).

In some embodiments, the TIL capable of secreting at least one, two, three, four, or five times or more IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least more than one fold of IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least more than twice IFN- γ is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least more than three times IFN- γ is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least four times more IFN- γ is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least more than five times IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D).

In some embodiments, a TIL capable of secreting at least 100pg/mL to about 1000pg/mL or more of IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 200pg/mL, at least 250pg/mL, at least 300pg/mL, at least 350pg/mL, at least 400pg/mL, at least 450pg/mL, at least 500pg/mL, at least 550pg/mL, at least 600pg/mL, at least 650pg/mL, at least 700pg/mL, at least 750pg/mL, at least 800pg/mL, at least 850pg/mL, at least 900pg/mL, at least 950pg/mL, or at least 1000pg/mL, or more, of IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 200pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 200pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 300pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 400pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 500pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 600pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 700pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 800pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 900pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 1000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 2000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 3000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 4000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 5000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 6000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 7000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 8000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 9000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 10,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 15,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 20,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 25,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 30,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 35,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 40,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 45,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 50,000pg/mL IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D).

In some embodiments, a TIL capable of secreting at least 100pg/mL/5e5 cells to about 1000pg/mL/5e5 cells or more IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the method is capable of secreting at least 200pg/mL/5e5 cells, at least 250pg/mL/5e5 cells, at least 300pg/mL/5e5 cells, at least 350pg/mL/5e5 cells, at least 400pg/mL/5e5 cells, at least 450pg/mL/5e5 cells, at least 500pg/mL/5e5 cells, at least 550pg/mL/5e5 cells, at least 600pg/mL/5e5 cells, at least 650pg/mL/5e5 cells, at least 700pg/mL/5e5 cells, at least 750pg/mL/5e5 cells, at least 800pg/mL/5e5 cells, at least 850pg/mL/5e5 cells, at least 900pg/mL/5e5 cells, at least 950pg/mL/5 cells, or at least 1000pg/mL/5e5 cells or more TIL is an expanded TIL (FIG. 8 and FIG. 8 or FIG. 8) of the invention and the method of producing TIB (e.g.g. 8 and FIG. 8). In some embodiments, a TIL capable of secreting at least 200pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 200pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 300pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 400pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 500pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 600pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 700pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 800pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 900pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 1000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 2000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 3000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 4000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 5000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 6000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 7000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 8000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 9000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 10,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 15,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 20,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 25,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 30,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 35,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 40,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 45,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 50,000pg/mL/5e5 cells IFN- γ is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D).

The various antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene segments: v (variable region), D (variable region), J (junction region) and C (constant region) determine the binding specificity and downstream application of immunoglobulins to T Cell Receptors (TCRs). The present invention provides a method for producing TIL that exhibits and increases T cell reservoir diversity. In some embodiments, the TIL obtained by the methods of the invention exhibits increased T cell reservoir diversity. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell depot diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those implemented in fig. 8 (e.g., particularly fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, the TIL obtained by the methods of the invention exhibits increased T cell depot diversity compared to freshly collected TIL and/or TIL prepared using a method referred to as Gen 2 as exemplified in fig. 8 (e.g., in particular fig. 8A). In some embodiments, the TIL obtained in the first expansion exhibits increased T cell reservoir diversity. In some embodiments, increasing diversity is increasing immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in the immunoglobulin and in the heavy chain of the immunoglobulin. In some embodiments, the diversity is present in the immunoglobulin and in the immunoglobulin light chain. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T Cell Receptor (TCR) α and/or β is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, TCRab (i.e., tcra/β) is expressed in increased. In some embodiments, a process as described herein (e.g., the Gen 3 process) exhibits higher clonal diversity based on the number of unique peptide CDRs within a sample as compared to other processes (e.g., the process known as Gen 2).

In some embodiments, activation and depletion of TIL may be determined by examining more than one marker. In some embodiments, activation and depletion can be determined using polychromatic flow cytometry. In some embodiments, activation and depletion of the label includes (but is not limited to) one or more labels selected from the group consisting of: CD3, PD-1, 2B4/CD244, CD8, CD25, BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183, CD69, CD95, CD127, CD103 and/or LAG-3). In some embodiments, activation and depletion of the label includes (but is not limited to) one or more labels selected from the group consisting of: BTLA, CTLA-4, ICOS, ki67, LAG-3, PD-1, TIGIT and/or TIM-3. In some embodiments, activation and depletion of the label includes (but is not limited to) one or more labels selected from the group consisting of: BTLA, CTLA-4, ICOS, ki67, LAG-3, CD103+/CD69+, CD103+/CD69-, PD-1, TIGIT and/or TIM-3. In some embodiments, T cell markers (including activation and depletion markers) can be determined and/or analyzed to examine T cell activation, inhibition, or function. In some embodiments, T cell markers may include (but are not limited to) one or more markers selected from the group consisting of: TIGIT, CD3, foxP3, tim-3, PD-1, CD103, CTLA-4, LAG-3, BTLA-4, ICOS, ki67, CD8, CD25, CD45, CD4 and/or CD59.

In some embodiments, TILs exhibiting secretion of greater than 3000pg/10 ⁶ TILs to 300000pg/10 ⁶ TILs or more of granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments of the present invention, in some embodiments, exhibit secretion of greater than 3000pg/10 ⁶ TILs, greater than 5000pg/10 ⁶ TILs, greater than 7000pg/10 ⁶ TILs, greater than 9000pg/10 ⁶ TILs, greater than 11000pg/10 ⁶ TILs, greater than 13000pg/10 ⁶ TILs, greater than 15000pg/10 ⁶ TILs, greater than 17000pg/10 ⁶ TILs, greater than 19000pg/10 ⁶ TILs, greater than 20000pg/10 ⁶ TILs, greater than 40000pg/10 ⁶ TILs, greater than 60000pg/10 ⁶ TILs, greater than 80000pg/10 ⁶ TILs, greater than 100000pg/10 ⁶ TILs TILs above 120000pg/10 ⁶ TILs, above 140000pg/10 ⁶ TILs, above 160000pg/10 ⁶ TILs, above 180000pg/10 ⁶ TILs, above 200000pg/10 ⁶ TILs, above 220000pg/10 ⁶ TILs, above 240000pg/10 ⁶ TILs, above 260000pg/10 ⁶ TILs, above 280000pg/10 ⁶ TILs, above 300000pg/10 ⁶ TILs or more of granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 3000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 5000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 7000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 9000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 11000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 13000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 15000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 17000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 19000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 20000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 40000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 60000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 80000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 100000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 120000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 140000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 160000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 180000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 200000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 220000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 240000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 260000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 280000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 300000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 3000pg/10 ⁶ TILs to 300000pg/10 ⁶ TILs or more of granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments of the present invention, in some embodiments, exhibit secretion of greater than 3000pg/10 ⁶ TILs, greater than 5000pg/10 ⁶ TILs, greater than 7000pg/10 ⁶ TILs, greater than 9000pg/10 ⁶ TILs, greater than 11000pg/10 ⁶ TILs, greater than 13000pg/10 ⁶ TILs, greater than 15000pg/10 ⁶ TILs, greater than 17000pg/10 ⁶ TILs, greater than 19000pg/10 ⁶ TILs, greater than 20000pg/10 ⁶ TILs, greater than 40000pg/10 ⁶ TILs, greater than 60000pg/10 ⁶ TILs, greater than 80000pg/10 ⁶ TILs, greater than 100000pg/10 ⁶ TILs TILs above 120000pg/10 ⁶ TILs, above 140000pg/10 ⁶ TILs, above 160000pg/10 ⁶ TILs, above 180000pg/10 ⁶ TILs, above 200000pg/10 ⁶ TILs, above 220000pg/10 ⁶ TILs, above 240000pg/10 ⁶ TILs, above 260000pg/10 ⁶ TILs, above 280000pg/10 ⁶ TILs, above 300000pg/10 ⁶ TILs or more of granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 3000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 5000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 7000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 9000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 11000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 13000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 15000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 17000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 19000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 20000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 40000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 60000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 80000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 100000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 120000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 140000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 160000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 180000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 200000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 220000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 240000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 260000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 280000pg/10 ⁶ TIL granzyme B are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 300000pg/10 ⁶ TIL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D).

In some embodiments, a TIL exhibiting secretion of more than 1000pg/mL to 300000pg/mL or more of granzyme B is a TIL produced by an amplification method of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TILs exhibiting secretion of greater than 1000pg/mL, greater than 2000pg/mL, greater than 3000pg/mL, greater than 4000pg/mL, greater than 5000pg/mL, greater than 6000pg/mL, greater than 7000pg/mL, greater than 8000pg/mL, greater than 9000pg/mL, greater than 10000pg/mL, greater than 20000pg/mL, greater than 30000pg/mL, greater than 40000pg/mL, greater than 50000pg/mL, greater than 60000pg/mL, greater than 70000pg/mL, greater than 80000pg/mL, greater than 90000pg/mL, greater than 100000pg/mL, or greater than 100000pg/mL are TILs produced by the amplification methods of the invention (including, e.g., fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL exhibiting greater than 1000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 2000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 3000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 4000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 5000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 6000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 7000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 8000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 9000pg/mL granzyme B is a TIL produced by an amplification method of the present invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 10000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting above 20000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 30000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 40000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 50000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 60000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 70000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 80000pg/mL granzyme B is a TIL produced by the amplification methods of the present invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 90000pg/mL granzyme B is a TIL produced by an amplification method of the present invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting greater than 100000pg/mL granzyme B is a TIL produced by an amplification method of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, a TIL exhibiting secretion of greater than 120000pg/mL granzyme B is a TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 140000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 160000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of above 180000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of above 200000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 220000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 240000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 260000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 280000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D). In some embodiments, TIL exhibiting secretion of greater than 300000pg/mL granzyme B is TIL produced by the amplification methods of the invention (including, e.g., fig. 8A and/or fig. 8B and/or fig. 8C and/or fig. 8D).

In some embodiments, the amplification methods of the invention produce an amplified TIL population exhibiting increased in vitro granzyme B secretion compared to a non-amplified TIL population, including, for example, TIL as provided in fig. 8A and/or 8B and/or 8C and/or 8D. In some embodiments, the amplified TIL population of the present invention has at least a one-fold to fifty-fold or more increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, IFN- γ secretion is increased by at least one fold, at least two fold, at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least eight fold, at least nine fold, at least ten fold, at least twenty fold, at least thirty fold, at least forty fold, at least fifty fold, or more compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a doubling of granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a two-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a three-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a four-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a five-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a six-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least seven-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least an eight-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a nine-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a tenfold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least twenty-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a thirty-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a forty-fold increase in granzyme B secretion compared to a non-amplified TIL population. In some embodiments, the amplified TIL population of the present invention has at least a fifty-fold increase in granzyme B secretion compared to a non-amplified TIL population.

In some embodiments, the TIL capable of secreting at least one, two, three, four, or five times or more lower levels of TNF-alpha (i.e., TNF-alpha) as compared to IFN-gamma secretion is the TIL produced by the amplification methods of the invention (including, e.g., the methods of FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D). In some embodiments, the TIL capable of secreting at least one fold lower levels of TNF- α as compared to IFN- γ secretion is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the TIL capable of secreting at least twice as much TNF- α as compared to IFN- γ secretion is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the TIL capable of secreting TNF- α at least three times lower than IFN- γ secretion is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the TIL capable of secreting TNF- α at least four times lower than IFN- γ secretion is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, the TIL capable of secreting TNF- α at least five times lower than IFN- γ secretion is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D).

In some embodiments, a TIL capable of secreting at least 200pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF- α (i.e., TNF-alpha) is a TIL produced by an amplification method of the invention (including, for example, the methods of FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D). In some embodiments, a TIL capable of secreting at least 500pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 1000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 2000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 3000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 4000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 5000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 6000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 7000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 8000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, a TIL capable of secreting at least 9000pg/mL/5e5 cells to about 10,000pg/mL/5e5 cells or more TNF-a is a TIL produced by an amplification method of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D).

In some embodiments, IFN- γ and granzyme B content is measured to determine the phenotypic characteristics of TIL produced by the amplification methods of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, IFN-gamma and TNF-alpha content are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the present invention, including, for example, the methods of FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D. In some embodiments, granzyme B and TNF- α content are measured to determine the phenotypic characteristics of TIL produced by the amplification methods of the invention (including, for example, the methods of fig. 8A and/or 8B and/or 8C and/or 8D). In some embodiments, IFN- γ, granzyme B, and TNF- α content are measured to determine the phenotypic characteristics of TILs produced by the amplification methods of the invention (including, for example, the methods of FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D).

In some embodiments, the phenotypic characteristic is checked after cryopreservation.

G. further process embodiment

In some embodiments, the invention provides a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) Obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Initiating a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein initiating the first amplification is performed for about 1 to 7 days or about 1 to 8 days to obtain a second population of TILs, the second population of TILs being greater in number than the first population of TILs; (c) Performing a rapid second expansion by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs), producing a third population of TILs, wherein the rapid second expansion is performed for about 1 to 11 days or about 1 to 10 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs; (d) Collecting the therapeutic TIL population obtained from step (c), and (e) genetically editing at least a portion of the TIL cells at any time before or after step (d) to express at least one immunomodulatory composition on the surface of the TIL cells. In some embodiments, the step of rapidly second amplifying is split into multiple steps to achieve a longitudinal expansion of the culture scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) effecting transfer of the second TIL population from the small-scale culture to a second vessel (e.g., G-REX-500MCS vessel) that is larger than the first vessel, wherein in the second vessel the second TIL population from the small-scale culture is cultured in the larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a first small-scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small-scale culture into at least 2,3,4, 5, 6,7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of equal size to the first container, in each of which second containers the portion of the second TIL population from the first small-scale culture transferred to such second container is cultured in the second small-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days or about 2 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small-scale culture into at least 2,3,4, 5, 6,7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which second containers the portion of the second TIL population transferred from the small-scale culture to such second container is cultured in the larger-scale culture for a period of about 4 to 7 days or about 4 to 8 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small scale culture into at least 2,3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which the second TIL population portion transferred from the small scale culture to such second containers is cultured in the larger scale culture for a period of about 5 to 7 days. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

In some embodiments, the invention provides methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) Obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Initiating a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein initiating the first amplification is performed for about 1 to 8 days to obtain a second population of TILs, the number of second population of TILs being greater than the first population of TILs; (c) Performing a rapid second expansion by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs), producing a third population of TILs, wherein the rapid second expansion is performed for about 1 to 8 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs; (d) Collecting the therapeutic TIL population obtained from step (c), and (e) genetically editing at least a portion of the TIL cells at any time before or after step (d) to express at least one immunomodulatory composition on the surface of the TIL cells. In some embodiments, the step of rapidly second amplifying is split into multiple steps to achieve a longitudinal expansion of the culture scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 2 to 4 days; and then (2) effecting transfer of the second TIL population from the small-scale culture to a second container (e.g., G-REX-500MCS container) that is larger than the first container, wherein in the second container the second TIL population from the small-scale culture is cultured in the larger-scale culture for a period of about 4 to 8 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a first small-scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 2 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of equal size to the first container, in each of which second containers the portion of the second TIL population from the first small-scale culture transferred to such second container is cultured in the second small-scale culture for a period of about 4 to 6 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 2 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which second containers the portion of the second TIL population transferred from the small-scale culture to such second container is cultured in the larger-scale culture for a period of about 4 to 6 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small scale culture into at least 2, 3, or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which the second TIL population portion transferred from the small scale culture to such second containers is cultured in the larger scale culture for a period of about 4 to 5 days. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

In some embodiments, the invention provides a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) Obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Initiating a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein initiating the first amplification is performed for about 1 to 7 days to obtain a second population of TILs, the second population of TILs being greater in number than the first population of TILs; (c) Performing a rapid second expansion by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs), producing a third population of TILs, wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs; (d) Collecting the therapeutic TIL population obtained from step (c), and (e) genetically editing at least a portion of the TIL cells at any time before or after step (d) to express at least one immunomodulatory composition on the surface of the TIL cells. In some embodiments, the step of rapidly second amplifying is split into multiple steps to achieve a longitudinal expansion of the culture scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days; and then (2) effecting transfer of the second TIL population from the small-scale culture to a second container (e.g., G-REX-500MCS container) that is larger than the first container, wherein in the second container the second TIL population from the small-scale culture is cultured in the larger-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a first small-scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of equal size to the first container, in each of which second containers the portion of the second TIL population from the first small-scale culture transferred to such second container is cultured in the second small-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 3 to 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which second containers the portion of the second TIL population transferred from the small-scale culture to such second container is cultured in the larger-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapidly expanding is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX100MCS vessel) for a period of about 4 days; and then (2) effecting transfer and partitioning of the second TIL population from the first small scale culture into at least 2, 3 or 4 second containers (e.g., G-REX-G500MCS containers) of larger size than the first container, in each of which the second TIL population portion transferred from the small scale culture to such second containers is cultured in the larger scale culture for a period of about 5 days. In some embodiments, at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first amplification is performed by contacting the first TIL population with a medium further comprising exogenous Antigen Presenting Cells (APCs), wherein the number of APCs in the medium in step (c) is greater than the number of APCs in the medium in step (b).

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c) the medium is supplemented with additional exogenous APCs.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 20:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 10:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 9:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 8:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 7:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 6:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 5:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 4:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 3:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 10:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 5:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 4:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 3:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.8:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.7:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.6:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.5:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.4:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.3:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or about 2:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or about 1.1:1、1.2:1、1.3:1、1.4:1、1.5:1、1.6:1、1.7:1、1.8:1、1.9:1、2:1、2.1:1、2.2:1、2.3:1、2.4:1、2.5:1、2.6:1、2.7:1、2.8:1、2.9:1、3:1、3.1:1、3.2:1、3.3:1、3.4:1、3.5:1、3.6:1、3.7:1、3.8:1、3.9:1、4:1、4.1:1、4.2:1、4.3:1、4.4:1、4.5:1、4.6:1、4.7:1、4.8:1、4.9:1 or 5:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the number of APCs added in the primary first amplification is exactly or about 1×10⁸、1.1×10⁸、1.2×10⁸、1.3×10⁸、1.4×10⁸、1.5×10⁸、1.6×10⁸、1.7×10⁸、1.8×10⁸、1.9×10⁸、2×10⁸、2.1×10⁸、2.2×10⁸、2.3×10⁸、2.4×10⁸、2.5×10⁸、2.6×10⁸、2.7×10⁸、2.8×10⁸、2.9×10⁸、3×10⁸、3.1×10⁸、3.2×10⁸、3.3×10⁸、3.4×10⁸ or 3.5X10 ⁸ APCs and the number of APCs added in the rapid second amplification is exactly or about 3.5×10⁸、3.6×10⁸、3.7×10⁸、3.8×10⁸、3.9×10⁸、4×10⁸、4.1×10⁸、4.2×10⁸、4.3×10⁸、4.4×10⁸、4.5×10⁸、4.6×10⁸、4.7×10⁸、4.8×10⁸、4.9×10⁸、5×10⁸、5.1×10⁸、5.2×10⁸、5.3×10⁸、5.4×10⁸、5.5×10⁸、5.6×10⁸、5.7×10⁸、5.8×10⁸、5.9×10⁸、6×10⁸、6.1×10⁸、6.2×10⁸、6.3×10⁸、6.4×10⁸、6.5×10⁸、6.6×10⁸、6.7×10⁸、6.8×10⁸、6.9×10⁸、7×10⁸、7.1×10⁸、7.2×10⁸、7.3×10⁸、7.4×10⁸、7.5×10⁸、7.6×10⁸、7.7×10⁸、7.8×10⁸、7.9×10⁸、8×10⁸、8.1×10⁸、8.2×10⁸、8.3×10⁸、8.4×10⁸、8.5×10⁸、8.6×10⁸、8.7×10⁸、8.8×10⁸、8.9×10⁸、9×10⁸、9.1×10⁸、9.2×10⁸、9.3×10⁸、9.4×10⁸、9.5×10⁸、9.6×10⁸、9.7×10⁸、9.8×10⁸、9.9×10⁸ or 1X 10 ⁹ APCs.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the number of APCs added in the primary first amplification is selected from the range of exactly or about 1×10 ⁸ APCs to exactly or about 3.5×10 ⁸ APCs and the number of APCs added in the rapid second amplification is selected from the range of exactly or about 3.5×10 ⁸ APCs to exactly or about 1×10 ⁹ APCs.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the number of APCs added in the primary first amplification is selected from the range of exactly or about 1.5×10 ⁸ APCs to exactly or about 3×10 ⁸ APCs and the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4×10 ⁸ APCs to exactly or about 7.5×10 ⁸ APCs.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the number of APCs added in the primary first amplification is selected from the range of exactly or about 2x 10 ⁸ APCs to exactly or about 2.5 x 10 ⁸ APCs and the number of APCs added in the rapid second amplification is selected from the range of exactly or about 4.5 x 10 ⁸ APCs to exactly or about 5.5 x 10 ⁸ APCs.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein exactly or about 2.5X10 ⁸ APCs are added to the primary first amplification and exactly or about 5X 10 ⁸ APCs are added to the rapid second amplification.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the plurality of tumor fragments are distributed into a plurality of separate containers, in each of which a first population of TILs is obtained in step (a), a second population of TILs is obtained in step (b), a third population of TILs is obtained in step (c), and the therapeutic populations of TILs from the plurality of containers in step (c) are pooled to produce a collected population of TILs from step (d).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of tumors are evenly distributed into a plurality of separate containers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of separate containers comprises at least two separate containers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the plurality of separate containers comprises two to twenty separate containers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the plurality of separate containers comprises two to fifteen separate containers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the plurality of separate containers comprises two to ten separate containers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of separate containers comprises two to five separate containers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of separate containers comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate containers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first population of TILs in step (b) is subjected to an initial first amplification in each vessel and the second population of TILs resulting from such first population of TILs is subjected to a rapid second amplification in step (c) in the same vessel.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each of the separate containers comprises a first gas permeable surface region.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of tumor fragments are distributed in a single container.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the single container comprises a first gas permeable surface region.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), and in step (c) the number of APCs added is greater than the number of APCs added in step (b), and in step (b) APCs are layered onto the first gas permeable surface region at an average thickness of from just or about one cell layer to just or about three cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b), the APC is layered onto the first gas permeable surface region with an average thickness of from just or about 1.5 cell layers to just or about 2.5 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b), the APC is layered onto the first gas permeable surface region with an average thickness of exactly or about 2 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b), APC is layered onto the first breathable surface zone with an average thickness of just or about: 1. 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered onto the first gas permeable surface region with an average thickness of from just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered onto the first gas permeable surface region with an average thickness of from just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (c) the APC is layered onto the first gas permeable surface region with an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered onto the first gas permeable surface region at an average thickness of about or below ：4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9 or 8 cell layers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein in step (b) the initial first amplification is performed in a first vessel comprising a first gas permeable surface region and in step (c) the rapid second amplification is performed in a second vessel comprising a second gas permeable surface region.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the second container is larger than the first container.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and in step (b) APCs are layered onto the first gas permeable surface region at an average thickness of from just or about one cell layer to just or about three cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable, wherein in step (b), APC is layered onto the first breathable surface zone with an average thickness of just or about: 1. 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered onto the second gas permeable surface region with an average thickness of from just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered onto the second gas permeable surface region with an average thickness of from just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (c) the APC is layered onto the second gas permeable surface region with an average thickness of just or about 3, 4,5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered onto the second gas permeable surface region with an average thickness of about or less than ：4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9 or 8 cell layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the initial first amplification is performed in a first vessel comprising a first gas permeable surface region, and in step (c) the rapid second amplification is performed in the first vessel.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:9.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from the range of exactly or about 1:2.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (b) the primary first expansion is performed by supplementing the cell culture medium of the first TIL population with additional Antigen Presenting Cells (APCs), the number of APCs added in step (c) being greater than the number of APCs added in step (b), the ratio of the average number of APCs stacked in step (b) to the average number of APCs stacked in step (c) being selected from just or about 1:1.1、1:1.2、1:1.3、1:1.4、1:1.5、1:1.6、1:1.7、1:1.8、1:1.9、1:2、1:2.1、1:2.2、1:2.3、1:2.4、1:2.5、1:2.6、1:2.7、1:2.8、1:2.9、1:3、1:3.1、1:3.2、1:3.3、1:3.4、1:3.5、1:3.6、1:3.7、1:3.8、1:3.9、1:4、1:4.1、1:4.2、1:4.3、1:4.4、1:4.5、1:4.6、1:4.7、1:4.8、1:4.9、1:5、1:5.1、1:5.2、1:5.3、1:5.4、1:5.5、1:5.6、1:5.7、1:5.8、1:5.9、1:6、1:6.1、1:6.2、1:6.3、1:6.4、1:6.5、1:6.6、1:6.7、1:6.8、1:6.9、1:7、1:7.1、1:7.2、1:7.3、1:7.4、1:7.5、1:7.6、1:7.7、1:7.8、1:7.9、1:8、1:8.1、1:8.2、1:8.3、1:8.4、1:8.5、1:8.6、1:8.7、1:8.8、1:8.9、1:9、1:9.1、1:9.2、1:9.3、1:9.4、1:9.5、1:9.6、1:9.7、1:9.8、1:9.9 or 1:10.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is from exactly or about 1.5:1 to exactly or about 100:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 50:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 25:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 20:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 10:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the second TIL population is at least just or about 50 times greater in number than the first TIL population.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the second TIL population is at least just or about 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49 or 50 times greater in number than the first TIL population.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the cell culture medium is supplemented with additional IL-2 just or about 2 days or just or about 3 days after the start of the second time period in step (c).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, further comprising the step of cryopreserving the collected population of TILs in step (d) by a cryopreservation process.

In other embodiments, the invention provides a modified method as described in any preceding paragraph applicable above, comprising the further step (e) of transferring the collected TIL population from step (d) to an infusion bag optionally containing HypoThermosol, after step (d).

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, comprising the step of cryopreserving the infusion bag containing the collected TIL population in step (e) by a cryopreservation process.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the cryopreservation process is performed using a 1:1 ratio of the collected TIL population to the cryopreservation media.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the PBMCs are irradiated and allogeneic.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (b) is 2.5 x 10 ⁸.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the total number of APCs added to the cell culture in step (c) is 5 x 10 ⁸.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the APC is PBMC.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the antigen presenting cells are artificial antigen presenting cells.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the collecting in step (d) is performed using a membrane-based cell processing system.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the collecting in step (d) is performed using a LOVO cell processing system.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 5 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 10 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 15 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from just or about 20 to just or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 25 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 30 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 35 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 40 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 45 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises exactly or about 50 to exactly or about 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises exactly or about 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59 or 60 fragments per container in step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of just or about 27mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of from just or about 20mm ³ to just or about 50mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of from just or about 21mm ³ to just or about 30mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of from just or about 22mm ³ to just or about 29.5mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of from just or about 23mm ³ to just or about 29mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of from just or about 24mm ³ to just or about 28.5mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of from just or about 25mm ³ to just or about 28mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of from just or about 26.5mm ³ to just or about 27.5mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each fragment has a volume of just or about: 21. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises from exactly or about 30 to exactly or about 60 fragments, wherein the total volume is from exactly or about 1300mm ³ to exactly or about 1500mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises exactly or about 50 fragments, in total volume of exactly or about 1350mm ³.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the plurality of fragments comprises exactly or about 50 fragments, with a total mass of exactly or about 1 gram to exactly or about 1.5 grams.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the cell culture medium is provided in a G-vessel or a container of Xuri cell bags.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the concentration of IL-2 in the cell culture medium is from about 10,000iu/mL to about 5,000iu/mL.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the concentration of IL-2 in the cell culture medium is about 6,000IU/mL.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the cryopreservation medium comprises Dimethylsulfoxide (DMSO).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the cryopreservation medium comprises 7% to 10% DMSO.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein the first time period in step (b) is performed over a time period of exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the second period of time in step (c) is performed over a period of time of exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first period of time in step (b) and the second period of time in step (c) are each performed over a period of time of exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, respectively.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first period of time in step (b) and the second period of time in step (c) are each performed over a period of time of exactly or about 5 days, 6 days, or 7 days, respectively.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first time period in step (b) and the second time period in step (c) are each performed within a time period of exactly or about 7 days, respectively.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of from just or about 14 days to just or about 18 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of from exactly or about 15 days to exactly or about 18 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 16 days to exactly or about 18 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 17 days to exactly or about 18 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of from just or about 14 days to just or about 17 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of from exactly or about 15 days to exactly or about 17 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 16 days to exactly or about 17 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of from just or about 14 days to just or about 16 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of from exactly or about 15 days to exactly or about 16 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 14 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 15 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 16 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 17 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for exactly or about 18 days in total.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of just or about 14 days or less.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of just or about 15 days or less.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed in total for just or about 16 days or less.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) through (d) are performed for a total of just or about 18 days or less.

In other embodiments, the invention provides a modified method as described in any preceding paragraph applicable above, wherein the population of therapeutic TILs collected in step (d) comprises a therapeutically effective dose of TIL sufficient for TIL.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the number of TILs sufficient for a therapeutically effective dose is from just or about 2.3 x 10 ¹⁰ to just or about 13.7 x 10 ¹⁰.

In other embodiments, the invention provides a modified method as described in any preceding paragraph above applies wherein the third TIL population in step (c) provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the third population of TILs in step (c) provides at least one to five times or more interferon-gamma production compared to TILs prepared by a process longer than 16 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the third population of TILs in step (c) provides at least one to five times or more interferon-gamma production compared to TILs prepared by a process longer than 17 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the third population of TILs in step (c) provides at least one to five times or more interferon-gamma production compared to TILs prepared by a process longer than 18 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the effector T cells and/or central memory T cells obtained from the third TIL population of step (c) exhibit increased CD8 and CD28 expression relative to the effector T cells and/or central memory T cells obtained from the second population of cells of step (b).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein each container recited in the method is a closed container.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each container recited in the method is a G container.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each container recited in the method is GREX-10.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each container recited in the method is GREX-100.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein each container recited in the method is GREX-500.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein at least one immunomodulatory composition comprises a membrane anchor fused to an immunomodulatory agent selected from the group consisting of ：IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、IL-23、IL-27、IL-4、IL-1α、IL-1β、IL-5、IFNγ、TNFα(TNFa)、IFNα、IFNβ、GM-CSF、GCSF、CD40 agonists (e.g., CD40L or agonistic CD40 binding domain) and biologically active variants thereof. In some embodiments, the immunomodulator is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immunomodulator is selected from: IL-12, IL-15, IL-18, IL-21 and CD40 agonists. In some embodiments, the immune modulator is IL-12. In some embodiments, the immunomodulator is IL-15. In some embodiments, the immunomodulator is IL-18. In some embodiments, the immunomodulator is IL-21. In some embodiments, the immunomodulator is a CD40 agonist (e.g., CD40L or an agonistic CD40 binding domain).

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the gene editor is a transient gene editor, wherein the transient gene editor TIL transiently expresses the at least one immunomodulatory composition. In some embodiments, the immunomodulatory composition comprises an immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises a membrane anchor fused to an immunomodulatory agent. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the cytokine is selected from: IL-2, IL-7, IL-10, IL-12, IL-15, IL-18 and IL-21. In some embodiments, the cytokine is selected from: IL-2, IL-12, IL-15, IL-18 and IL-21. In some embodiments, the cytokine is selected from: IL-12, IL-15, IL-18 and IL-21. In some embodiments, the cytokine is IL-12. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokine is IL-21. In some embodiments, the immunomodulator is a CD40 agonist (e.g., CD40L or an agonistic CD40 binding domain).

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared by a method as described in any preceding paragraph applicable above.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from tumor tissue of a patient, wherein a plurality of cells in the population of therapeutic TILs comprise at least one immunomodulatory composition on the cell surface, as compared to TILs prepared by a process in which first expansion of TILs is performed in the absence of any added Antigen Presenting Cells (APCs) or OKT 3.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the population of therapeutic TILs provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process in which first expansion of TILs is performed in the absence of any added Antigen Presenting Cells (APCs); the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the population of therapeutic TILs provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process in which first amplification of TILs is performed in the absence of any added OKT 3; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the population of therapeutic TILs provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality as compared to TILs prepared by a process in which a first expansion of TILs is performed in the absence of added Antigen Presenting Cells (APCs) and in the absence of added OKT 3; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the population of therapeutic TILs provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process longer than 16 days; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the population of therapeutic TILs provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process longer than 17 days; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the population of therapeutic TILs provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process longer than 18 days; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface.

In other embodiments, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs as applicable above, which therapeutic TIL population provides increased interferon-gamma production.

In other embodiments, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs as applicable above, which therapeutic TIL population provides increased polyclonality.

In other embodiments, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs as applicable above, which therapeutic TIL population provides increased efficacy.

In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least one fold more interferon-gamma than TILs prepared by a process longer than 16 days. In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least one fold more interferon-gamma than TILs prepared by a process longer than 17 days. In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least one fold more interferon-gamma than TILs prepared by a process longer than 18 days. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least more than one fold of interferon- γ.

In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least more than twice as much interferon-gamma than TILs prepared by a process longer than 16 days. In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least more than twice as much interferon-gamma than TILs prepared by a process longer than 17 days. In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least more than twice as much interferon-gamma than TILs prepared by a process longer than 18 days. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least two times more interferon- γ.

In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least more than three times more interferon-gamma than TILs prepared by a process longer than 16 days. In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least more than three times more interferon-gamma than TILs prepared by a process longer than 17 days. In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above, wherein the population of therapeutic TILs is capable of producing at least more than three times more interferon-gamma than TILs prepared by a process longer than 18 days. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least three times more interferon- γ.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) capable of producing at least more than one fold of interferon-gamma compared to TILs prepared by a process in which the first expansion of TILs is performed in the absence of any added Antigen Presenting Cells (APCs); the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least more than one fold of interferon- γ.

In other embodiments, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) capable of producing at least more than one fold of interferon-gamma compared to TILs prepared by a process in which the first expansion of TILs is performed in the absence of any added OKT 3; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least more than one fold of interferon- γ.

In other embodiments, the invention provides a therapeutic TIL population capable of producing at least more than twice as much interferon-gamma as compared to a TIL prepared by a process in which a first amplification of TIL is performed without any added APC; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least two times more interferon- γ.

In other embodiments, the invention provides a therapeutic TIL population capable of producing at least more than twice as much interferon-gamma than a TIL prepared by a process in which a first amplification of TIL is performed in the absence of any added OKT 3; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface. In some embodiments, due to the amplification procedures described herein, e.g., in steps a to F, or according to steps a to F, herein (as also shown, e.g., in fig. 8 (particularly, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least two times more interferon- γ.

In other embodiments, the invention provides a therapeutic TIL population capable of producing at least more than three times more interferon-gamma than a TIL prepared by a process in which a first amplification of TIL is performed without any added APC; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least more than one fold of interferon- γ.

In other embodiments, the invention provides a therapeutic TIL population capable of producing at least more than three times more interferon-gamma than a TIL prepared by a process in which a first amplification of TIL is performed in the absence of any added OKT 3; the plurality of cells in the therapeutic TIL population includes at least one immunomodulatory composition on the cell surface. In some embodiments, due to the amplification process described herein, e.g., in steps a to F above or according to steps a to F above (as also shown, e.g., in fig. 8 (in particular, e.g., fig. 8A and/or 8B and/or 8C and/or 8D)), TIL is capable of producing at least three times more interferon- γ.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy), a coarse biopsy, a core biopsy, or a fine needle aspirate.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the tumor fragments are coarse needle biopsy.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the tumor fragments are fine needle aspirates.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a perforated biopsy).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the tumor fragment is a core needle biopsy.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs adapted as applicable above such that (i) the method comprises obtaining a first population of TILs from more than one small biopsy (including, for example, a perforated biopsy), a coarse biopsy, a core biopsy, or a fine needle aspirate of tumor tissue from a subject; (ii) The method comprises the following steps prior to performing the initial first amplification step: culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days; (iii) The method comprises performing a first amplification for a period of about 8 days; and (iv) the method comprises performing a rapid second amplification for a period of about 11 days. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above such that (i) the method comprises obtaining a first population of TILs from more than one small biopsy (including, for example, a perforated biopsy), a coarse needle biopsy, a core needle biopsy, or a fine needle aspirate from tumor tissue of a subject; (ii) The method comprises the following steps prior to performing the initial first amplification step: culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days; (iii) The method comprises performing a first amplification for a period of about 8 days; and (iv) the method comprises performing a rapid second amplification by: culturing the culture of the second TIL population for about 5 days, dividing the culture into up to 5 subcultures, and culturing the subculture for about 6 days. In some of the foregoing embodiments, up to 5 subcultures are separately cultured in a vessel of the same size or larger than the vessel in which the second TIL population was initially cultured in the rapid second amplification. In some of the foregoing embodiments, cultures of the second TIL population are averaged over up to 5 subcultures. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs adapted as applicable above such that the first TIL population is obtained from 1 to about 20 small biopsy (including, for example, a perforated biopsy), a coarse needle biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue from a subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs adapted as applicable above such that the first TIL population is obtained from 1 to about 10 small biopsy (including, for example, a perforated biopsy), a coarse needle biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue from a subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs adapted as above such that the first TIL population is obtained from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 small biopsy (including, for example, a perforated biopsy), a coarse needle biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue from a subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs adapted as applicable above such that the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 small biopsy (including, for example, a perforated biopsy), a coarse needle biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue from a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1 to about 20 coarse needle biopsy of tumor tissue from a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1 to about 10 coarse needle biopsy of tumor tissue from a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 crude needle biopsy of tumor tissue from a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 crude needle biopsy of tumor tissue from a subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs that is modified as applicable above such that the first TIL population is obtained from 1 to about 20 fine needle aspirates from tumor tissue of the subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs that is modified as applicable above such that the first TIL population is obtained from 1 to about 10 fine needle aspirates from tumor tissue of the subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs that is modified as applicable above such that the first TIL population is obtained from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fine needle aspirates from tumor tissue of the subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs that is modified as applicable above such that the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fine needle aspirates from tumor tissue of the subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1 to about 20 core needle biopsy from tumor tissue of a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1 to about 10 core needle biopsy from tumor tissue of a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsy from tumor tissue of a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1,2, 3, 4,5, 6, 7, 8, 9, or 10 core needle biopsy from tumor tissue of the subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1 to about 20 small biopsy (including, for example, perforated biopsy) of tumor tissue from a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1 to about 10 small biopsy (including, for example, perforated biopsy) of tumor tissue from a subject.

In other embodiments, the invention provides a method as described in any of the preceding paragraphs that is modified as applicable above such that the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 small biopsy (including, for example, a perforated biopsy) of tumor tissue from a subject.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above such that the first TIL population is obtained from 1,2, 3,4,5,6, 7, 8, 9, or 10 small biopsy (including, for example, a perforated biopsy) of tumor tissue from a subject.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, such that (i) the method comprises obtaining a first population of TILs from 1 to about 10 coarse needle biopsy of tumor tissue from a subject; (ii) The method comprises the following steps prior to performing the initial first amplification step: culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days; (iii) The method comprises performing an initial first expansion step by culturing a first population of TILs in a cell culture medium comprising IL-2, OKT-3 and Antigen Presenting Cells (APCs) for a period of about 8 days to obtain a second population of TILs; (iv) The method comprises performing a rapid second amplification step by culturing the second TIL population in a cell culture medium comprising IL-2, OKT-3 and APC for a period of about 11 days. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, such that (i) the method comprises obtaining a first population of TILs from 1 to about 10 coarse needle biopsy of tumor tissue from a subject; (ii) The method comprises the following steps prior to performing the initial first amplification step: culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days; (iii) The method comprises performing an initial first expansion step by culturing a first population of TILs in a cell culture medium comprising IL-2, OKT-3 and Antigen Presenting Cells (APCs) for a period of about 8 days to obtain a second population of TILs; (iv) The method comprises performing a rapid second amplification by: culturing the culture of the second TIL population in a cell culture medium comprising IL-2, OKT-3 and APC for about 5 days, dividing the culture into up to 5 subcultures, and culturing each of these subcultures in a cell culture medium comprising IL-2 for about 6 days. In some of the foregoing embodiments, up to 5 subcultures are separately cultured in a vessel of the same size or larger than the vessel in which the second TIL population was initially cultured in the rapid second amplification. In some of the foregoing embodiments, cultures of the second TIL population are averaged over up to 5 subcultures. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph applicable above, wherein (i) the method comprises obtaining a first population of TILs from 1 to about 10 crude needle biopsy of tumor tissue from a subject; (ii) The method comprises the following steps prior to performing the initial first amplification step: culturing the first TIL population in a G-REX-100M flask in a cell culture medium comprising 6000IU IL-2/mL of 0.5L CM1 medium for a period of about 3 days; (iii) The method comprises initiating a first amplification by: adding 0.5L of CM1 medium containing 6000IU/mL IL-2, 30ng/mL OKT-3 and about 10 ⁸ feeder cells, and culturing for a period of about 8 days; (iv) The method comprises performing a rapid second amplification by: (a) Transferring the second TIL population to a G-REX-500MCS flask containing 5L CM2 medium with 3000IU/mL IL-2, 30ng/mL OKT-3, and 5X 10 ⁹ feeder cells, and culturing for about 5 days; (b) Cultures were divided into up to 5 subcultures by transferring 10 ⁹ TILs into each of up to 5G-REX-500 MCS flasks containing 5L AIM-V medium with 3000IU/mL IL-2, and the subcultures were cultured for about 6 days. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the invention provides a method of expanding T cells comprising: (a) Performing an initial first expansion of a first T cell population obtained from the donor by culturing the first T cell population to effect growth and initiate activation of the first T cell population; (b) After the activation of the first T cell population initiated in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population to effect growth and enhance activation of the first T cell population, obtaining a second T cell population; (c) collecting a second population of T cells; and (d) genetically editing a portion of the T cell to express at least one immunomodulatory composition on the surface of the T cell at any time before or after step (c). In other embodiments, the step of rapid second amplification is split into multiple steps to achieve a longitudinal expansion of the culture scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days; and (b) effecting transfer of the first T cell population from the small-scale culture to a second vessel (e.g., a G-REX-500MCS vessel) that is larger than the first vessel, culturing the first T cell population from the small-scale culture in the larger-scale culture in the second vessel for a period of about 4 to 7 days. In other embodiments, the rapid amplification step is split into multiple steps to achieve lateral expansion of the culture scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a first small-scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the first small scale culture into at least 2,3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second vessels of larger size than the first vessel, in each of which the portion of the first T cell population from the first small scale culture transferred to such second vessel is cultured in the second small scale culture for a period of about 4 to 7 days. In other embodiments, the rapid expansion step is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the small scale culture into at least 2,3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which the portion of the first T cell population from the small scale culture transferred to such second container is cultured in the larger scale culture for a period of about 4 to 7 days. In other embodiments, the rapid expansion step is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 4 days; and (b) effecting transfer and distribution of the first T cell population from the small-scale culture into at least 2,3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first containers, in each of which the portion of the first T cell population from the small-scale culture transferred to such second containers is cultured in the larger-scale culture for a period of about 5 days.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulator is selected from: IL-2, IL-12, IL-15, IL-18, IL-21 and CD40 agonists (e.g., CD40L or agonistic CD40 binding domain).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the step of rapidly second amplifying is split into a plurality of steps to achieve a longitudinal expansion of the culture scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 2 to 4 days; and (b) effecting transfer of the first T cell population from the small-scale culture to a second vessel (e.g., a G-REX-500MCS vessel) that is larger than the first vessel, culturing the first T cell population from the small-scale culture in the larger-scale culture in the second vessel for a period of about 5 to 7 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the step of rapidly expanding is split into a plurality of steps to achieve lateral expansion of the culture scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a first small-scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 2 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the first small-scale culture into at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers of equal size to the first container, in each of which the portion of the first T cell population from the first small-scale culture transferred to such second container is cultured in the second small-scale culture for a period of about 5 to 7 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the step of rapidly expanding is split into a plurality of steps to achieve culture scale-up laterally and scale-up longitudinally by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 2 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the small scale culture into at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500MCS containers) of larger size than the first container, in each of which the portion of the first T cell population from the small scale culture transferred to such second container is cultured in the larger scale culture for a period of about 5 to 7 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the step of rapidly expanding is split into a plurality of steps to achieve culture scale-up laterally and scale-up longitudinally by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the small-scale culture into 2, 3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first containers, in each of which the portion of the first T cell population from the small-scale culture transferred to such second containers is cultured in the larger-scale culture for a period of about 5 to 6 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the rapid expansion step is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the small-scale culture into 2,3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first containers, in each of which the portion of the first T cell population from the small-scale culture transferred to such second containers is cultured in the larger-scale culture for a period of about 5 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the rapid expansion step is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the small-scale culture into 2,3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first containers, in each of which the portion of the first T cell population from the small-scale culture transferred to such second containers is cultured in the larger-scale culture for a period of about 6 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the rapid expansion step is split into multiple steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale by: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days; and (b) effecting transfer and distribution of the first T cell population from the small-scale culture into 2,3 or 4 second containers (e.g., G-REX-500MCS containers) of larger size than the first containers, in each of which the portion of the first T cell population from the small-scale culture transferred to such second containers is cultured in the larger-scale culture for a period of about 7 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the initial first amplification of step (a) is performed for a period of up to 7 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed for a period of up to 8 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the rapid second amplification of step (b) is performed for a period of up to 9 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the rapid second amplification of step (b) is performed for a period of up to 10 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the rapid second amplification of step (b) is performed for a period of up to 11 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the initial first amplification in step (a) is performed for a period of 7 days and the rapid second amplification in step (b) is performed for a period of up to 9 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the initial first amplification in step (a) is performed for a period of 7 days and the rapid second amplification in step (b) is performed for a period of up to 10 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the initial first amplification in step (a) is performed for a period of 7 days or 8 days and the rapid second amplification in step (b) is performed for a period of up to 9 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the initial first amplification in step (a) is performed for a period of 7 days or 8 days and the rapid second amplification in step (b) is performed for a period of up to 10 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the initial first amplification in step (a) is performed for a period of 8 days and the rapid second amplification in step (b) is performed for a period of up to 9 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the initial first amplification in step (a) is performed for a period of 8 days and the rapid second amplification of step (b) is performed for a period of up to 8 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein, in step (a), the first T cell population is cultured in a first medium comprising OKT-3 and IL-2.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the first medium comprises a 4-1BB agonist, OKT-3 and IL-2.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the first medium comprises OKT-3, IL-2 and Antigen Presenting Cells (APCs).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the first medium comprises a 4-1BB agonist, OKT-3, IL-2 and Antigen Presenting Cells (APC).

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein, in step (b), the first T cell population is cultured in a second medium comprising OKT-3, IL-2 and Antigen Presenting Cells (APCs).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the second medium comprises a 4-1BB agonist, OKT-3, IL-2 and Antigen Presenting Cells (APC).

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of T cells is cultured in a first medium comprising OKT-3, IL-2 and a first population of Antigen Presenting Cells (APCs) in a container comprising a first gas permeable surface, the first population of APCs being exogenous to a donor of the first population of T cells, the first population of APCs being layered onto the first gas permeable surface, and in step (b) the first population of T cells is cultured in a second medium comprising OKT-3, IL-2 and a second population of APCs, the second population of APCs being exogenous to a donor of the first population of T cells, the second population of APCs being layered onto the first gas permeable surface, the second population of APCs being larger than the first population of APCs.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of T cells is cultured in a first medium comprising a 4-1BB agonist, OKT-3, IL-2, and a first population of Antigen Presenting Cells (APCs) in a container comprising a first gas permeable surface, the first population of APCs being exogenous to a first population of T cells donor, the first population of APCs being layered onto the first gas permeable surface, and in step (b) the first population of T cells is cultured in a second medium comprising OKT-3, IL-2, and a second population of APCs, the second population of APCs being exogenous to a donor of the first population of T cells, the second population of APCs being layered onto the first gas permeable surface, the second population of APCs being larger than the first population of APCs.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of T cells is cultured in a first medium comprising OKT-3, IL-2 and a first population of Antigen Presenting Cells (APCs) in a container comprising a first gas permeable surface, the first population of APCs being exogenous to a donor of the first population of T cells, the first population of APCs being layered onto the first gas permeable surface, and in step (b) the first population of T cells is cultured in a second medium comprising a 4-1BB agonist, OKT-3, IL-2 and a second population of APCs, the second population of APCs being exogenous to a donor of the first population of APCs, the second population of APCs being layered onto the first gas permeable surface, the second population of APCs being larger than the first population of APCs.

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of T cells is cultured in a first medium comprising a 4-1BB agonist, OKT-3, IL-2, and a first population of Antigen Presenting Cells (APCs) in a container comprising a first gas permeable surface, the first population of APCs being exogenous to a first population of T cells donor, the first population of APCs being layered onto the first gas permeable surface, and in step (b) the first population of T cells is cultured in a second medium comprising a 4-1BB agonist, OKT-3, IL-2, and a second population of APCs, the second population being exogenous to a donor of the first population of T cells, the second population of APCs being layered onto the first gas permeable surface, the second population of APCs being larger than the first population of APCs.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the ratio of the number of APCs in the second population of APCs to the number of APCs in the first population of APCs is about 2:1.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the number of APCs in the first population of APCs is about 2.5×10 ⁸ and the number of APCs in the second population of APCs is about 5×10 ⁸.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (a) the first population of APCs is layered onto the first ventilation surface at an average thickness of 2 APC layers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the second population of APCs is layered onto the first air permeable surface with an average thickness in the range selected from 4 to 8 APC layers.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the ratio of the average number of APC layers laminated onto the first ventilation surface in step (b) to the average number of APC layers laminated onto the first ventilation surface in step (a) is 2:1.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (a) the first population of APCs is seeded on the first gas permeable surface at a density selected from the range of just or about 1.0 x 10 ⁶ APCs/cm ² to just or about 4.5 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (a) the first population of APCs is seeded on the first gas permeable surface at a density selected from the range of just or about 1.5 x 10 ⁶ APCs/cm ² to just or about 3.5 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (a) the first population of APCs is seeded on the first gas permeable surface at a density selected from the range of just or about 2.0 x 10 ⁶ APCs/cm ² to just or about 3.0 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (a) the first population of APCs is seeded onto the first gas permeable surface at a density of exactly or about 2.0 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the second population of APCs is seeded on the first gas permeable surface at a density selected from the range of just or about 2.5 x 10 ⁶ APCs/cm ² to just or about 7.5 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the second population of APCs is seeded on the first gas permeable surface at a density selected from the range of just or about 3.5 x 10 ⁶ APCs/cm ² to just or about 6.0 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the second population of APCs is seeded on the first gas permeable surface at a density selected from the range of just or about 4.0 x 10 ⁶ APCs/cm ² to just or about 5.5 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the second population of APCs is seeded onto the first gas permeable surface at a density of exactly or about 4.0 x 10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of APCs is seeded onto the first air permeable surface at a density selected from the range of just or about 1.0×10 ⁶ APCs/cm ² to just or about 4.5×10 ⁶ APCs/cm ², and in step (b) the second population of APCs is seeded onto the first air permeable surface at a density selected from the range of just or about 2.5×10 ⁶ APCs/cm ² to just or about 7.5×10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of APCs is seeded onto the first air permeable surface at a density selected from the range of just or about 1.5×10 ⁶ APCs/cm ² to just or about 3.5×10 ⁶ APCs/cm ², and in step (b) the second population of APCs is seeded onto the first air permeable surface at a density selected from the range of just or about 3.5×10 ⁶ APCs/cm ² to just or about 6.0×10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of APCs is seeded onto the first air permeable surface at a density selected from the range of just or about 2.0×10 ⁶ APCs/cm ² to just or about 3.0×10 ⁶ APCs/cm ², and in step (b) the second population of APCs is seeded onto the first air permeable surface at a density selected from the range of just or about 4.0×10 ⁶ APCs/cm ² to just or about 5.5×10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any preceding paragraph where applicable above, wherein in step (a) the first population of APCs is seeded onto the first ventilation surface at a density of just or about 2.0×10 ⁶ APCs/cm ², and in step (b) the second population of APCs is seeded onto the first ventilation surface at a density of just or about 4.0×10 ⁶ APCs/cm ².

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the APCs are Peripheral Blood Mononuclear Cells (PBMCs).

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the PBMCs are irradiated and exogenous to the donor of the first T cell population.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the T cells are Tumor Infiltrating Lymphocytes (TILs).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the T cells are bone Marrow Infiltrating Lymphocytes (MILs).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the T cells are Peripheral Blood Lymphocytes (PBLs).

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained by whole blood isolation from a donor.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained by isolation of a apheresis product from the donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the first T cell population is isolated from whole blood or apheresis products of the donor by positive or negative selection of T cell phenotypes.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs applicable above, wherein the T cell phenotype is cd3+ and cd45+.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the T cells are isolated from the NK cells prior to performing the initial first expansion of the first T cell population. In other embodiments, T cells in the first T cell population are separated from NK cells by removing CD3-cd56+ cells from the first T cell population. In other embodiments, the CD3-cd56+ cells are removed from the first T cell population by cell sorting the first T cell population using a gating strategy that removes the CD3-cd56+ cell fraction and recovers the negative fraction. In other embodiments, the foregoing methods are used for T cell expansion in a first T cell population characterized by a high percentage of NK cells. In other embodiments, the foregoing methods are used for T cell expansion in a first T cell population characterized by a high percentage of CD3-CD56+ cells. In other embodiments, the foregoing methods are used for T cell expansion in tumor tissue characterized by the presence of large numbers of NK cells. In other embodiments, the foregoing methods are used for T cell expansion in tumor tissue characterized by a large number of CD3-CD56+ cells. In other embodiments, the foregoing methods are used for T cell expansion in tumor tissue obtained from a patient having a tumor characterized by the presence of a large number of NK cells. In other embodiments, the foregoing methods are used for T cell expansion in tumor tissue obtained from a patient having a tumor characterized by the presence of a plurality of CD3-cd56+ cells. In other embodiments, the foregoing methods are used for T cell expansion in tumor tissue obtained from a patient suffering from ovarian cancer.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein exactly or about 1 x 10 ⁷ T cells from the first T cell population are seeded in a vessel to initiate primary first expansion culture in such vessel.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first population of T cells is distributed into a plurality of containers, and exactly or about 1 x 10 ⁷ T cells from the first population of T cells are seeded in each container to initiate primary first expansion culture in such containers.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the second population of T cells collected in step (c) is a therapeutic population of TILs.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from one or more small biopsy (including, for example, a perforated biopsy), a coarse needle biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsy of tumor tissue from a donor (including, for example, a perforated biopsy), a coarse biopsy, a core biopsy, or a fine needle aspirate.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsy (including, for example, a perforated biopsy), a coarse needle biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1,2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 small biopsies of tumor tissue from a donor (including, for example, perforated biopsies), coarse needle biopsies, core needle biopsies or fine needle aspirates.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1,2, 3, 4,5, 6, 7, 8, 9 or 10 small biopsies of tumor tissue from a donor (including, for example, a perforated biopsies), a coarse needle biopsies, a core needle biopsies or a fine needle aspirate.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from one or more rough needle biopsy of tumor tissue from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 tumor tissue coarse needle biopsy from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 tumor tissue coarse needle biopsy from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1,2,3, 4,5, 6, 7,8,9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue coarse needle biopsy from the donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1,2, 3, 4, 5, 6, 7,8, 9 or 10 crude needle biopsy of tumor tissue from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from more than one tumor tissue fine needle aspirate from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 tumor tissue fine needle aspirates from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 tumor tissue fine needle aspirates from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue fine needle aspirates from the donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 tumor tissue fine needle aspirates from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from one or more small biopsies of tumor tissue from a donor (including, for example, perforated biopsies).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies of tumor tissue from the donor (including, for example, perforated biopsies).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies of tumor tissue from the donor (including, for example, perforated biopsies).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 small biopsies of tumor tissue from a donor (including, for example, perforated biopsies).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1, 2,3, 4,5, 6,7, 8, 9 or 10 small biopsies of tumor tissue from a donor (including, for example, perforated biopsies).

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from one or more tumor tissue core needle biopsy from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 20 tumor tissue core needle biopsy from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1 to 10 tumor tissue core needle biopsy from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1,2,3, 4,5, 6,7, 8,9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tumor tissue core needle biopsies from a donor.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first T cell population is obtained from 1,2,3,4, 5, 6, 7, 8, 9 or 10 tumor tissue core needle biopsy from a donor.

In other embodiments, the invention provides methods of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: i) Obtaining and/or receiving a first TIL population from a tumor sample obtained from one or more small, coarse or penetrating biopsy of a tumor of a subject by culturing the tumor sample in a first cell culture medium comprising IL-2 for about 3 days; (ii) Initiating a first expansion by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed in a vessel having a first gas permeable surface region, the initiating the first expansion being performed for a first period of about 7 or 8 days to obtain the second population of TILs, the second population of TILs being greater in number than the first population of TILs; (iii) Generating a third population of TILs by performing a rapid second amplification by supplementing a second cell culture medium of the second population of TILs with additional IL-2, OKT-3 and APCs, wherein the number of APCs added in the rapid second amplification is at least twice the number of APCs added in step (ii), the rapid second amplification being performed for a second period of about 11 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs, the rapid second amplification being performed in a container having a second gas permeable surface area; (iv) Collecting the therapeutic TIL population obtained from step (iii); (v) Transferring the collected TIL population from step (iv) to an infusion bag; and (vi) genetically editing at least a portion of the TIL cells at any time before or after step (iv) to express at least one immunomodulatory composition on the surface of the TIL cells.

In other embodiments, the invention provides methods of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: i) Obtaining and/or receiving a first TIL population from a tumor sample obtained from one or more small, coarse or penetrating biopsy of a tumor of a subject by culturing the tumor sample in a first cell culture medium comprising IL-2 for about 3 days; (ii) Initiating a first expansion by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed for a first period of time of about 7 or 8 days to obtain the second population of TILs, the number of the second population of TILs being greater than the first population of TILs; (iii) Performing a rapid second amplification by contacting the second population of TILs with a third cell culture medium comprising IL-2, OKT-3, and APC, producing a third population of TILs, wherein the rapid second amplification is performed for a second period of about 11 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs, the rapid second amplification being performed in a vessel having a second gas permeable surface region; (iv) Collecting the therapeutic TIL population obtained from step (iii); and (v) genetically editing at least a portion of the TIL cells at any time before or after step (iv) to express at least one immunomodulatory composition on the surface of the TIL cells.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein after day 5 of the second time period, the culture is divided into 2 or more subcultures, each subculture is supplemented with an additional amount of the third medium and cultured for about 6 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein after day 5 of the second period of time, the culture is divided into 2 or more subcultures, each subculture is supplemented with a fourth medium comprising IL-2 and cultured for about 6 days.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the culture is divided into up to 5 subcultures after day 5 of the second period.

In other embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein all steps in the method are completed within about 22 days.

In other embodiments, the invention provides a method of expanding T cells comprising: (i) Performing an initial first expansion of the first T cell population by culturing the first T cell population to effect growth and initiate activation of the first T cell population, the tumor sample obtained from one or more small, coarse needle, or penetrating biopsy of the donor tumor; (ii) After the activation of the first T cell population initiated in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population to effect growth and enhance activation of the first T cell population, obtaining a second T cell population; (iv) collecting a second population of T cells; and (v) genetically editing at least a portion of the T cells to express at least one immunomodulatory composition on the surface of the T cells at any time before or after step (iv). In some embodiments, the tumor sample is obtained from a plurality of coarse needle biopsy sections. In some embodiments, the plurality of coarse needle biopsy slices is selected from: 2.3, 4, 5, 6, 7, 8, 9 and 10 coarse needle biopsy.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein at least one immunomodulatory composition comprises a membrane anchor fused to an immunomodulatory agent selected from the group consisting of ：IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、IL-23、IL-27、IL-4、IL-1α、IL-1β、IL-5、IFNγ、TNFα(TNFa)、IFNα、IFNβ、GM-CSF、GCSF、CD40 agonists (e.g., CD40L or agonistic CD40 binding domain) and biologically active variants thereof. In some embodiments, the cytokine is selected from: IL-12, IL-15, IL-18 and IL-21. In some embodiments, the cytokine is IL-12. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokine is IL-21. In some embodiments, the immunomodulator is a CD40 agonist (e.g., CD40L or an agonistic CD40 binding domain).

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the T cells or TILs are obtained from tumor digests. In some embodiments, tumor digests are produced by incubating the tumor in an enzyme medium (such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10mg/mL gentamicin, 30U/mL dnase, and 1.0mg/mL collagenase), followed by mechanical dissociation (GENTLEMACS of the biotechnology company, obumeday, california). In some embodiments, the tumor is placed in a tumor dissociating enzyme mixture comprising one or more dissociating (digesting) enzymes, such as, but not limited to, collagenase (including any mixture or type of collagenase), accutase ^TM、Accumax^TM, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, proteinase type XIV (chain protease), deoxyribonuclease I (dnase), trypsin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof. In other embodiments, the tumor is placed in a tumor dissociating enzyme mixture comprising collagenase (including collagenase of any mixture or type), neutral protease (dispase), and dnase I (dnase).

IX. pharmaceutical compositions, dosages and dosing regimens

In some embodiments, the TIL, MILs, or PBLs (including TIL, MILs, or PBLs genetically modified to express CCR) amplified and/or genetically modified using the methods of the present disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TIL amplified using PBMCs of the present disclosure may be administered by any suitable route known in the art. In some embodiments, the T cells are administered in the form of a single intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable dose of TIL may be administered. In some embodiments, about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs are administered, with an average of about 7.8×10 ¹⁰ TILs, particularly where the cancer is NSCLC or melanoma. In some embodiments, about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs are administered. In some embodiments, about 3 x 10 ¹⁰ to about 12 x 10 ¹⁰ TILs are administered. In some embodiments, about 4 x 10 ¹⁰ to about 10 x 10 ¹⁰ TILs are administered. In some embodiments, about 5 x 10 ¹⁰ to about 8 x 10 ¹⁰ TILs are administered. In some embodiments, about 6 x 10 ¹⁰ to about 8 x 10 ¹⁰ TILs are administered. In some embodiments, about 7 x 10 ¹⁰ to about 8 x 10 ¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is from about 2.3 x 10 ¹⁰ to about 13.7 x 10 ¹⁰. In some embodiments, the therapeutically effective dose is about 7.8x10 ¹⁰ TILs, particularly where the cancer is melanoma. In some embodiments, the therapeutically effective dose is about 7.8x10 ¹⁰ TILs, particularly where the cancer is NSCLC. In some embodiments, the therapeutically effective dose is from about 1.2 x 10 ¹⁰ to about 4.3 x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 3 x 10 ¹⁰ to about 12 x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 4 x 10 ¹⁰ to about 10 x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 5 x 10 ¹⁰ to about 8 x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 6 x 10 ¹⁰ to about 8 x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 7 x 10 ¹⁰ to about 8 x 10 ¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1×10⁶、2×10⁶、3×10⁶、4×10⁶、5×10⁶、6×10⁶、7×10⁶、8×10⁶、9×10⁶、1×10⁷、2×10⁷、3×10⁷、4×10⁷、5×10⁷、6×10⁷、7×10⁷、8×10⁷、9×10⁷、1×10⁸、2×10⁸、3×10⁸、4×10⁸、5×10⁸、6×10⁸、7×10⁸、8×10⁸、9×10⁸、1×10⁹、2×10⁹、3×10⁹、4×10⁹、5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、1×10¹¹、2×10¹¹、3×10¹¹、4×10¹¹、5×10¹¹、6×10¹¹、7×10¹¹、8×10¹¹、9×10¹¹、1×10¹²、2×10¹²、3×10¹²、4×10¹²、5×10¹²、6×10¹²、7×10¹²、8×10¹²、9×10¹²、1×10¹³、2×10¹³、3×10¹³、4×10¹³、5×10¹³、6×10¹³、7×10¹³、8×10¹³ and 9 x 10 ¹³. In some embodiments, the number of TILs provided in the pharmaceutical compositions of the present invention is in the range of 1 x 10 ⁶ to 5 x 10 ⁶、5×10⁶ to 1 x 10 ⁷、1×10⁷ to 5 x 10 ⁷、5×10⁷ to 1 x 10 ⁸、1×10⁸ to 5 x 10 ⁸、5×10⁸ to 1 x 10 ⁹、1×10⁹ to 5 x 10 ⁹、5×10⁹ to 1 x 10 ¹⁰、1×10¹⁰ to 5 x 10 ¹⁰、5×10¹⁰ to 1 x 10 ¹¹、5×10¹¹ to 1 x 10 ¹²、1×10¹² to 5 x 10 ¹² and 5 x 10 ¹² to 1 x 10 ¹³.

In some embodiments, the concentration of TIL provided in the pharmaceutical compositions of the invention is less than 100％、90％、80％、70％、60％、50％、40％、30％、20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％、0.4％、0.3％、0.2％、0.1％、0.09％、0.08％、0.07％、0.06％、0.05％、0.04％、0.03％、0.02％、0.01％、0.009％、0.008％、0.007％、0.006％、0.005％、0.004％、0.003％、0.002％、0.001％、0.0009％、0.0008％、0.0007％、0.0006％、0.0005％、0.0004％、0.0003％、0.0002％ or 0.0001% w/w, w/v or v/v of the pharmaceutical composition, for example.

In some embodiments, the concentration of TIL provided in the pharmaceutical compositions of the invention is greater than 90％、80％、70％、60％、50％、40％、30％、20％、19.75％、19.50％、19.25％、19％、18.75％、18.50％、18.25％、18％、17.75％、17.50％、17.25％、17％、16.75％、16.50％、16.25％、16％、15.75％、15.50％、15.25％、15％、14.75％、14.50％、14.25％、14％、13.75％、13.50％、13.25％、13％、12.75％、12.50％、12.25％、12％、11.75％、11.50％、11.25％、11％、10.75％、10.50％、10.25％、10％、9.75％、9.50％、9.25％、9％、8.75％、8.50％、8.25％、8％、7.75％、7.50％、7.25％、7％、6.75％、6.50％、6.25％、6％、5.75％、5.50％、5.25％、5％、4.75％、4.50％、4.25％、4％、3.75％、3.50％、3.25％、3％、2.75％、2.50％、2.25％、2％、1.75％、1.50％、125％、1％、0.5％、0.4％、0.3％、0.2％、0.1％、0.09％、0.08％、0.07％、0.06％、0.05％、0.04％、0.03％、0.02％、0.01％、0.009％、0.008％、0.007％、0.006％、0.005％、0.004％、0.003％、0.002％、0.001％、0.0009％、0.0008％、0.0007％、0.0006％、0.0005％、0.0004％、0.0003％、0.0002％ or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of TIL provided in the pharmaceutical compositions of the present invention is in the range of about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of TIL provided in the pharmaceutical compositions of the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the amount of TIL provided in the pharmaceutical compositions of the present invention is equal to or less than 10g、9.5g、9.0g、8.5g、8.0g、7.5g、7.0g、6.5g、6.0g、5.5g、5.0g、4.5g、4.0g、3.5g、3.0g、2.5g、2.0g、1.5g、1.0g、0.95g、0.9g、0.85g、0.8g、0.75g、0.7g、0.65g、0.6g、0.55g、0.5g、0.45g、0.4g、0.35g、0.3g、0.25g、0.2g、0.15g、0.1g、0.09g、0.08g、0.07g、0.06g、0.05g、0.04g、0.03g、0.02g、0.01g、0.009g、0.008g、0.007g、0.006g、0.005g、0.004g、0.003g、0.002g、0.001g、0.0009g、0.0008g、0.0007g、0.0006g、0.0005g、0.0004g、0.0003g、0.0002g or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical compositions of the present invention is greater than 0.0001g、0.0002g、0.0003g、0.0004g、0.0005g、0.0006g、0.0007g、0.0008g、0.0009g、0.001g、0.0015g、0.002g、0.0025g、0.003g、0.0035g、0.004g、0.0045g、0.005g、0.0055g、0.006g、0.0065g、0.007g、0.0075g、0.008g、0.0085g、0.009g、0.0095g、0.01g、0.015g、0.02g、0.025g、0.03g、0.035g、0.04g、0.045g、0.05g、0.055g、0.06g、0.065g、0.07g、0.075g、0.08g、0.085g、0.09g、0.095g、0.1g、0.15g、0.2g、0.25g、0.3g、0.35g、0.4g、0.45g、0.5g、0.55g、0.6g、0.65g、0.7g、0.75g、0.8g、0.85g、0.9g、0.95g、1g、1.5g、2g、2.5、3g、3.5、4g、4.5g、5g、5.5g、6g、6.5g、7g、7.5g、8g、8.5g、9g、9.5g or 10g.

TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The exact dosage will depend on the route of administration, the form of administration of the compound, the sex and age of the subject to be treated, the weight of the subject to be treated, and the preferences and experience of the attending physician. Clinically determined doses of TIL may also be used as appropriate. The amount of pharmaceutical composition administered using the methods herein, e.g., the dosage of TIL, will depend on the severity of the human or mammal, disorder or condition being treated, the rate of administration, the configuration of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

In some embodiments, the TIL may be administered in a single dose. Such administration may be by injection, such as intravenous injection. In some embodiments, the TIL may be administered in multiple doses. The administration may be once, twice, three times, four times, five times, six times or more than six times per year. Administration may be once a month, once every two weeks, once a week, or once every other day. Administration of the TIL may continue as desired.

In some embodiments, the effective dose of TIL is about 1×10⁶、2×10⁶、3×10⁶、4×10⁶、5×10⁶、6×10⁶、7×10⁶、8×10⁶、9×10⁶、1×10⁷、2×10⁷、3×10⁷、4×10⁷、5×10⁷、6×10⁷、7×10⁷、8×10⁷、9×10⁷、1×10⁸、2×10⁸、3×10⁸、4×10⁸、5×10⁸、6×10⁸、7×10⁸、8×10⁸、9×10⁸、1×10⁹、2×10⁹、3×10⁹、4×10⁹、5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、1×10¹¹、2×10¹¹、3×10¹¹、4×10¹¹、5×10¹¹、6×10¹¹、7×10¹¹、8×10¹¹、9×10¹¹、1×10¹²、2×10¹²、3×10¹²、4×10¹²、5×10¹²、6×10¹²、7×10¹²、8×10¹²、9×10¹²、1×10¹³、2×10¹³、3×10¹³、4×10¹³、5×10¹³、6×10¹³、7×10¹³、8×10¹³ and 9 x 10 ¹³. In some embodiments, the effective dose of TIL is in the range of 1×10 ⁶ to 5×10 ⁶、5×10⁶ to 1×10 ⁷、1×10⁷ to 5×10 ⁷、5×10⁷ to 1×10 ⁸、1×10⁸ to 5×10 ⁸、5×10⁸ to 1×10 ⁹、1×10⁹ to 5×10 ⁹、5×10⁹ to 1×10 ¹⁰、1×10¹⁰ to 5×10 ¹⁰、5×10¹⁰ to 1×10 ¹¹、5×10¹¹ to 1×10 ¹²、1×10¹² to 5×10 ¹² and 5×10 ¹² to 1×10 ¹³.

In some embodiments, the effective dose of TIL is in the range of about 0.01mg/kg to about 4.3mg/kg, about 0.15mg/kg to about 3.6mg/kg, about 0.3mg/kg to about 3.2mg/kg, about 0.35mg/kg to about 2.85mg/kg, about 0.15mg/kg to about 2.85mg/kg, about 0.3mg/kg to about 2.15mg/kg, about 0.45mg/kg to about 1.7mg/kg, about 0.15mg/kg to about 1.3mg/kg, about 0.3mg/kg to about 1.15mg/kg, about 0.45mg/kg to about 1mg/kg, about 0.55mg/kg to about 0.85mg/kg, about 0.65mg/kg to about 0.8mg/kg, about 0.7mg/kg to about 0.75mg/kg, about 0.7mg/kg to about 2.15mg/kg, about 2.15mg/kg to about 1.3mg/kg, about 1.3mg/kg to about 1.15mg/kg to about 1.3mg/kg, about 0.55mg/kg to about 1.85mg/kg, about 0.5 mg/kg to about 0.5 mg/kg, about 3.5 mg/kg to about 3.5 mg/kg.

In some embodiments, an effective dose of TIL is in a range of about 1mg to about 500mg, about 10mg to about 300mg, about 20mg to about 250mg, about 25mg to about 200mg, about 1mg to about 50mg, about 5mg to about 45mg, about 10mg to about 40mg, about 15mg to about 35mg, about 20mg to about 30mg, about 23mg to about 28mg, about 50mg to about 150mg, about 60mg to about 140mg, about 70mg to about 130mg, about 80mg to about 120mg, about 90mg to about 110mg, or about 95mg to about 105mg, about 98mg to about 102mg, about 150mg to about 250mg, about 160mg to about 240mg, about 170mg to about 230mg, about 180mg to about 220mg, about 190mg to about 210mg, about 195mg to about 205mg, or about 198 to about 207 mg.

An effective amount of TIL may be administered in single or multiple doses by any of the modes of acceptance of agents having similar utility, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, by implantation, or by inhalation.

In other embodiments, the invention provides an infusion bag comprising a therapeutic TIL population as described in any of the preceding paragraphs.

In other embodiments, the invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described in any preceding paragraph above and a pharmaceutically acceptable carrier.

In other embodiments, the invention provides an infusion bag comprising a TIL composition as described in any of the preceding paragraphs.

In other embodiments, the invention provides a cryopreserved formulation of a therapeutic TIL population as described in any preceding paragraph.

In other embodiments, the invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described in any preceding paragraph above and a cryopreservation medium.

In other embodiments, the invention provides a modified TIL composition as described in any preceding paragraph above, wherein the cryopreservation medium contains DMSO.

In other embodiments, the invention provides a modified TIL composition as described in any preceding paragraph above, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the invention provides a cryopreserved formulation of a TIL composition as described in any of the preceding paragraphs.

In some embodiments, TIL amplified using the methods of the present disclosure is administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TIL amplified using PBMCs of the present disclosure may be administered by any suitable route known in the art. In some embodiments, the T cells are administered in the form of a single intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable dose of TIL may be administered. In some embodiments, about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs are administered, with an average of about 7.8×10 ¹⁰ TILs, particularly where the cancer is NSCLC. In some embodiments, about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs are administered. In some embodiments, about 3x 10 ¹⁰ to about 12 x 10 ¹⁰ TILs are administered. In some embodiments, about 4 x 10 ¹⁰ to about 10x 10 ¹⁰ TILs are administered. In some embodiments, about 5 x 10 ¹⁰ to about 8x 10 ¹⁰ TILs are administered. In some embodiments, about 6 x 10 ¹⁰ to about 8x 10 ¹⁰ TILs are administered. In some embodiments, about 7 x 10 ¹⁰ to about 8x 10 ¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is from about 2.3 x 10 ¹⁰ to about 13.7 x 10 ¹⁰. In some embodiments, the therapeutically effective dose is about 7.8x10 ¹⁰ TILs, particularly the cancer is NSCLC. In some embodiments, the therapeutically effective dose is from about 1.2 x 10 ¹⁰ to about 4.3 x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 3x 10 ¹⁰ to about 12 x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 4 x 10 ¹⁰ to about 10x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 5 x 10 ¹⁰ to about 8x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 6 x 10 ¹⁰ to about 8x 10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 7 x 10 ¹⁰ to about 8x 10 ¹⁰ TILs.

TIL provided in the pharmaceutical compositions of the present invention is effective over a broad dosage range. The exact dosage will depend on the route of administration, the form of administration of the compound, the sex and age of the subject to be treated, the weight of the subject to be treated, and the preferences and experience of the attending physician. Clinically determined doses of TIL may also be used as appropriate. The amount of pharmaceutical composition administered using the methods herein, e.g., the dosage of TIL, will depend on the severity of the human or mammal, disorder or condition being treated, the rate of administration, the configuration of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

An effective amount of TIL may be administered in single or multiple doses by any of the modes of reception of agents with similar utility, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, by implantation, or by inhalation.

Method for treating patient

The treatment method starts with original TIL collection and TIL culture. Such methods are described, for example, in Jin et al, J.Immunotherapy, 2012,35 (3): 283-292, which is incorporated herein by reference in its entirety. Embodiments of the methods of treatment are described below throughout various sections, including examples.

The amplified TIL according to the methods described herein, including, for example, those described in steps A-F above or produced according to steps A-F above (as also shown, for example, in FIG. 1 and/or FIG. 8), find particular use in treating cancer patients (e.g., as described in Goff et al, J.Clinopodium Oncum, 2016,34 (20): 2389-239, and supplements, which are incorporated herein by reference in their entirety). In some embodiments, TIL is grown from resected metastatic melanoma stock as previously described (see Dudley et al, J.Immunotherapy, 2003,26:332-342, incorporated herein by reference in its entirety). Fresh tumors can be segmented under sterile conditions. Representative samples may be collected for formal pathology analysis. Individual fragments of 2mm ³ to 3mm ³ may be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 25, or 30 samples are obtained from each patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained from each patient. In some embodiments, 24 samples are obtained from each patient. Samples can be placed in individual wells of a 24-well plate, maintained in growth medium containing high doses of IL-2 (6,000IU/mL), and monitored for tumor destruction and/or TIL proliferation. Any tumor enzymes that remain viable cells after treatment can be digested into single cell suspensions and cryopreserved as described herein.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD 3, CD4, CD8, and CD 56) and tested against autologous tumors when available. TIL is considered reactive if the overnight co-culture yields an interferon-gamma (IFN-gamma) content of > 200pg/mL and twice background. (Goff et al, J.Immunotherapy, 2010,33:840-847; incorporated herein by reference in its entirety). In some embodiments, cultures that have been demonstrated to have an autoreactive or sufficient growth pattern may be selected for a second amplification (e.g., the second amplification provided in step D according to fig. 1 and/or fig. 8), including what is sometimes referred to as rapid amplification (REP). In some embodiments, amplified TILs with high self-reactivity (e.g., high proliferation during second amplification) are selected for additional second amplification. In some embodiments, TILs with high self-reactivity (e.g., high proliferation during the second amplification as provided in step D of fig. 1 and/or fig. 8) are selected for additional second amplification according to step D of fig. 1 and/or fig. 8.

The cell phenotype of the cryopreserved samples of infusion bags TIL can be analyzed by flow cytometry (e.g., flowJo) for surface markers CD3, CD4, CD8, CCR7, and CD45RA (bidi bioscience) as well as by any of the methods described herein. Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. The rise in serum IFN-g is defined as > 100pg/mL and a baseline level of greater than 43.

In some embodiments, the TIL produced by the methods provided herein, e.g., the methods illustrated in fig. 1 and/or 8, achieves a surprising improvement in clinical efficacy of the TIL. In some embodiments, TILs produced by methods provided herein, e.g., the methods illustrated in fig. 1 and/or 8, exhibit improved clinical efficacy compared to TILs produced by methods other than those described herein, including, e.g., methods other than those illustrated in fig. 1 and/or 8. In some embodiments, methods other than those described herein include methods known as process 1C and/or generation 1 (Gen 1). In some embodiments, increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by methods provided herein, such as the methods illustrated in fig. 1, exhibit similar reaction times and safety profiles as compared to TILs produced by methods other than those described herein, including, for example, methods other than those illustrated in fig. 1 and/or 8.

In some embodiments, IFN- γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN- γ in the blood of a subject treated with TIL is indicative of active TIL. In some embodiments, potency assays for IFN-gamma production are employed. IFN-gamma production is another measure of cytotoxic potential. IFN-y production may be measured by determining the level of the cytokine IFN-y in blood, serum or in isolated TIL of a subject treated with TIL prepared by the methods of the invention, including methods as described, for example, in FIG. 1 and/or FIG. 8. In some embodiments, an increase in IFN- γ indicates the efficacy of treatment of a patient treated with TIL produced by the methods of the invention. In some embodiments, IFN- γ is doubled, tripled, quadrupled, or quintupling or more compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 and/or fig. 8. In some embodiments, IFN- γ secretion is doubled compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 and/or fig. 8. In some embodiments, IFN- γ secretion is increased by a factor of two compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 and/or fig. 8. In some embodiments, IFN- γ secretion is increased by a factor of three compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 and/or fig. 8. In some embodiments, IFN- γ secretion is increased by a factor of four compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 and/or fig. 8. In some embodiments, IFN- γ secretion is increased five-fold compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 and/or fig. 8. In some embodiments, IFN-. Gamma.is measured using a Quantikine ELISA kit. In some embodiments, IFN- γ is measured in ex vivo TIL of a subject treated with a method of the invention, including TIL as prepared, for example, in the methods described in fig. 1 and/or fig. 8. In some embodiments, IFN- γ in the blood of a subject treated with a method of the invention, including TIL as prepared, for example, in the methods described in fig. 1 and/or fig. 8, is measured. In some embodiments, IFN- γ is measured in TIL serum of a subject treated with a method of the invention, including TIL as prepared, for example, in the methods described in fig. 1 and/or fig. 8. In some embodiments, IFN- γ indicates therapeutic efficacy and/or increased clinical efficacy in treating tumors.

In some embodiments, TILs prepared by the methods of the present invention include those as described, for example, in fig. 1. In some embodiments, IFN- γ indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN- γ in the blood of a subject treated with TIL is indicative of active TIL. In some embodiments, potency assays for IFN-gamma production are employed. IFN-gamma production is another measure of cytotoxic potential. IFN-y production may be measured by measuring the amount of the cytokine IFN-y in blood, serum or in isolated TIL of a subject treated with TIL prepared by the methods of the invention, including methods as described, for example, in FIG. 1 and/or FIG. 8. In some embodiments, an increase in IFN- γ indicates the efficacy of treatment of a patient treated with TIL produced by the methods of the invention. In some embodiments, IFN- γ is doubled, tripled, quadrupled, or five or more times IFN- γ compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than the methods provided herein, including, for example, methods other than those embodied in fig. 1 and/or fig. 8.

In some embodiments, TILs prepared by the methods of the present invention (including methods as described, for example, in fig. 1 and/or fig. 8) exhibit increased polyclonality compared to TILs produced by other methods (including methods not illustrated in fig. 1 and/or fig. 8, including methods such as the method referred to as the process 1C method). In some embodiments, significantly increased polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy. In some embodiments, polyclonality refers to T cell reservoir diversity. In some embodiments, an increase in polyclonality may be indicative of therapeutic efficacy with respect to administration of TIL produced by the methods of the present invention. In some embodiments, the polyclonality is increased 1-fold, 2-fold, 10-fold, 100-fold, 500-fold, or 1000-fold as compared to a TIL prepared using a method other than the methods provided herein, including, for example, a method other than the methods implemented in fig. 1 and/or fig. 8. In some embodiments, the polyclonality is increased by a factor of 1 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using methods other than those provided herein, including, for example, methods other than those implemented in fig. 1 and/or fig. 8. In some embodiments, the polyclonality is increased by a factor of 2 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using methods other than those provided herein, including, for example, methods other than those implemented in fig. 1 and/or fig. 8. In some embodiments, the polyclonality is increased by a factor of 10 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using methods other than those provided herein, including, for example, methods other than those implemented in fig. 1 and/or fig. 8. In some embodiments, the polyclonality is increased by a factor of 100 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using methods other than those provided herein, including, for example, methods other than those implemented in fig. 1 and/or fig. 8. In some embodiments, the polyclonality is increased 500-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in fig. 1 and/or fig. 8). In some embodiments, the polyclonality is increased 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those implemented in fig. 1 and/or fig. 8).

Metrics of efficacy may include Disease Control Rate (DCR) and total response rate (ORR), as known in the art and described herein.

A. methods of treating cancer

The compositions and methods described herein are useful in a method of treating a disease. In some embodiments, it is used to treat hyperproliferative disorders, such as cancer, in adult patients or pediatric patients. It may also be used to treat other conditions as described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from: anal cancer, bladder cancer, breast cancer (including triple negative breast cancer), bone cancer, cancer caused by Human Papilloma Virus (HPV), central nervous system related cancers (including ependymoma, neural blastoma, neuroblastoma, pineal blastoma and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer and cervical adenocarcinoma), colorectal cancer, endometrial cancer, esophageal cancer, cancer at the esophageal-gastric junction, gastric cancer, gastrointestinal stromal tumor, neuroglioblastoma, glioma, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC), laryngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma and pharyngeal carcinoma), renal cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including uveal melanoma, choriocarcinoma or iris melanoma), mesothelioma (including malignant mesothelioma), ovarian cancer, pancreatic cancer (including ductal adenocarcinoma), penile carcinoma, rectal cancer, renal sarcoma, carcinoma, sarcoma, osteosarcoma, carcinoma of the thyroid gland (including sarcoma, carcinoma of the uterus, and other fibrosarcoma, and carcinoma).

In some embodiments, the hyperproliferative disorder is a hematopoietic malignancy. In some embodiments, the hematopoietic malignancy is selected from: chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-hodgkin's lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the invention includes methods of treating a patient having cancer, which is a hematopoietic malignancy. In some embodiments, the invention includes methods of treating a patient with cancer, which is a hematopoietic malignancy, using a TIL, MILs, or PBL modified to express more than one CCR. In some embodiments, the invention includes methods of treating a patient with cancer, which is a hematopoietic malignancy, using MILs or PBLs modified to express more than one CCR.

In some embodiments, the cancer is one of the foregoing cancers, including solid tumor cancers and hematopoietic malignancies, that are recurrent or refractory to treatment with at least one prior therapy (including chemotherapy, radiation therapy, or immunotherapy). In some embodiments, the cancer is one of the foregoing cancers that is recurrent or refractory to treatment with at least two previous therapies (including chemotherapy, radiation therapy, and/or immunotherapy). In some embodiments, the cancer is one of the foregoing cancers that is recurrent or refractory to treatment with at least three previous therapies (including chemotherapy, radiation therapy, and/or immunotherapy).

In some embodiments, the cancer is a microsatellite instability high (MSI-H) or mismatch repair deficient cancer. MSI-H and dMMR cancers and their detection have been described in Kawakami et al, current oncology treatment options (Curr. Treat. Options Oncol.) 2015,16,30, the disclosure of which is incorporated herein by reference.

In some embodiments, the invention includes methods of treating a patient having cancer using a TIL, MILs, or PBL modified to express more than one CCR, wherein the patient is a human. In some embodiments, the invention includes methods of treating a patient with cancer using a TIL, MILs, or PBL modified to express more than one CCR, wherein the patient is non-human. In some embodiments, the invention includes methods of treating a patient having cancer using a TIL, MILs, or PBL modified to express more than one CCR, wherein the patient is a companion animal.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor and/or a MEK inhibitor. In some embodiments, the invention includes a method of treating a patient having cancer, wherein the cancer is refractory to a BRAF inhibitor selected from the group consisting of: vemurafenib (vemurafenib), dabrafenib (dabrafenib), enrafenib (encorafenib), sorafenib (sorafenib) and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the invention includes a method of treating a patient having a cancer, wherein the cancer is refractory to treatment with a MEK inhibitor selected from the group consisting of: trametinib (trametinib), cobimetinib, bemetinib (binimetinib), semetinib (selumetinib), pimecretinib (pimasertinib), refatinib (refametinib) and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the invention includes a method of treating a patient having cancer, wherein the cancer is refractory to a BRAF inhibitor selected from the group consisting of: vemurafenib, dabrafenib, enrafenib, sorafenib and pharmaceutically acceptable salts or solvates thereof; and are difficult to treat with MEK inhibitors selected from the group consisting of: trametinib, cobicitinib, bemetinib, semetinib, pimasemide, and refatinib, and pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the invention includes a method of treating a patient having a cancer, wherein the cancer is pediatric cancer.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the cancer is uveal melanoma.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the uveal melanoma is choroidal melanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the pediatric cancer is a neuroblastoma.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the pediatric cancer is a sarcoma.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the pediatric cancer is a Central Nervous System (CNS) -related cancer. In some embodiments, pediatric cancers are difficult to treat with chemotherapy. In some embodiments, pediatric cancers are difficult to treat with radiation therapy. In some embodiments, pediatric cancers are refractory to treatment with denominator (dinutuximab).

In some embodiments, the invention includes a method of treating a patient having cancer, wherein the CNS-related cancer is a neuroblastoma, pineal blastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein are useful in methods of treating cancer, wherein the cancer is refractory to treatment with an anti-PD-1 or anti-PD-L1 antibody or is resistant to pretreatment. In some embodiments, the patient is a primary refractory patient to an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient does not show a prior response to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient shows a previous response against PD-1 or an anti-PD-L1 antibody, followed by progression of the patient's cancer. In some embodiments, the cancer is refractory to treatment with an anti-CTLA-4 antibody and/or an anti-PD-1 or anti-PD-L1 antibody in combination with at least one chemotherapeutic agent. In some embodiments, the previous chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed (pemetrexed), and/or cisplatin. In some previous embodiments, the chemotherapeutic agent is a platinum dual chemotherapeutic agent. In some embodiments, the platinum dual therapy comprises a first chemotherapeutic agent selected from cisplatin and carboplatin, and a second chemotherapeutic agent selected from vinorelbine (vinorelbine), gemcitabine (gemcitabine), and a taxane including, for example, paclitaxel (paclitaxel), docetaxel (docetaxel), or albumin-bound paclitaxel (nab-paclitaxel). In some embodiments, the platinum dual chemotherapeutic agent is combined with pemetrexed.

In some embodiments, NSCLC is PD-L1 negative and/or from a patient with a cancer that expresses PD-L1 and has a Tumor Proportion Score (TPS) <1%, as described elsewhere herein.

In some embodiments, NSCLC is refractory to a combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody and a platinum dual therapy, wherein the platinum dual therapy comprises:

i) A first chemotherapeutic agent selected from cisplatin and carboplatin, and

Ii) a second chemotherapeutic agent selected from the group consisting of: vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel, docetaxel, or albumin-bound paclitaxel).

In some embodiments, NSCLC is refractory to a combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody, pemetrexed, and a platinum dual therapy, wherein the platinum dual therapy comprises:

i) A first chemotherapeutic agent selected from cisplatin and carboplatin, and

In some embodiments, NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient has not received treatment. In some embodiments, NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, NSCLC has been previously treated with a chemotherapeutic agent. In some embodiments, NSCLC has been previously treated with a chemotherapeutic agent, but is currently no longer treated with the chemotherapeutic agent. In some embodiments, the patient with NSCLC has not used anti-PD-1/PD-L1. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the patient with NSCLC is not NSCLC treated or has been treated with a chemotherapeutic agent, but is not PD-1/PD-L1 treated. In some embodiments, the NSCLC patient is untreated or post-chemotherapy treated, but is not anti-PD-1/PD-L1 treated and has low PD-L1 expression. In some embodiments, the NSCLC patient has a massive disease at baseline. In some embodiments, the subject has a bulk disease at baseline and has low PD-L1 expression. In some embodiments, the NSCLC patient does not have detectable PD-L1 expression. In some embodiments, a patient with NSCLC is untreated or treated with a chemotherapeutic agent but not treated with anti-PD-1/PD-L1 and has no detectable PD-L1 expression. In some embodiments, the patient has a large baseline at baseline and no detectable PD-L1 expression. In some embodiments, a patient with NSCLC has untreated NSCLC or is treated with chemotherapy (e.g., a chemotherapeutic agent) but is not treated with anti-PD-1/PD-L1, and the patient has low PD-L1 expression and/or has massive disease at baseline. In some embodiments, massive disease is indicated when the largest tumor diameter measured in the transverse or coronal plane is greater than 7 cm. In some embodiments, massive disease is indicated when there are swollen lymph nodes with a minor axis diameter of 20mm or more. In some embodiments, the chemotherapeutic agent comprises a standard-of-care therapeutic agent for NSCLC.

In some embodiments, PD-L1 expression is determined by tumor proportion scoring. In some embodiments, a subject with refractory NSCLC tumor has a Tumor Proportion Score (TPS) of < 1%. In some embodiments, a subject with refractory NSCLC tumor has ≡1% TPS. In some embodiments, a subject with refractory NSCLC has been previously treated with an anti-PD-1 and/or anti-PD-L1 antibody and a tumor proportion score has been determined prior to the anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject with refractory NSCLC has been previously treated with an anti-PD-L1 antibody and a tumor proportion score has been determined prior to the anti-PD-L1 antibody treatment.

In some embodiments, TILs prepared by the methods of the invention (including, for example, those methods as described in fig. 1 or 8) exhibit increased polyclonality compared to TILs produced by other methods (including those methods not illustrated in fig. 1 or 8, including, for example, methods known as the process 1C method). In some embodiments, significantly increased polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy of the cancer treatment. In some embodiments, polyclonality refers to T cell reservoir diversity. In some embodiments, an increase in polyclonality may be indicative of therapeutic efficacy with respect to administration of TIL produced by the methods of the present invention. In some embodiments, the polyclonality is increased 1-fold, 2-fold, 10-fold, 100-fold, 500-fold, or 1000-fold as compared to a TIL prepared using a method other than the methods provided herein (including, for example, a method other than the methods implemented in fig. 1 or 8). In some embodiments, the polyclonality is increased by a factor of 1 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than provided herein (including, for example, a method other than the method implemented in fig. 1 or fig. 8). In some embodiments, the polyclonality is increased by a factor of 2 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than provided herein (including, for example, a method other than the method implemented in fig. 1 or fig. 8). In some embodiments, the polyclonality is increased by a factor of 10 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than provided herein (including, for example, a method other than the method implemented in fig. 1 or fig. 8). In some embodiments, the polyclonality is increased by a factor of 100 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than provided herein (including, for example, a method other than the method implemented in fig. 1 or fig. 8). In some embodiments, the polyclonality is increased by a factor of 500 compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than provided herein (including, for example, a method other than the method implemented in fig. 1 or fig. 8). In some embodiments, the polyclonality is increased 1000-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than provided herein (including, for example, a method other than the method implemented in fig. 1 or fig. 8).

In some embodiments, PD-L1 expression is determined by tumor proportion scoring using one or more test methods as described herein. In some embodiments, a subject or patient with NSCLC tumor has a Tumor Proportion Score (TPS) of < 1%. In some embodiments, the NSCLC tumor has ≡1% TPS. In some embodiments, a subject or patient suffering from NSCLC has been previously treated with an anti-PD-1 and/or anti-PD-L1 antibody and a tumor proportion score has been determined prior to the anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject or patient with NSCLC has been previously treated with an anti-PD-L1 antibody and a tumor proportion score has been determined prior to the anti-PD-L1 antibody treatment. In some embodiments, a subject or patient with refractory or resistant NSCLC tumor has a Tumor Proportion Score (TPS) of < 1%. In some embodiments, a subject or patient with refractory or resistant NSCLC tumor has ≡1% TPS. In some embodiments, a subject or patient with refractory or resistant NSCLC has been previously treated with an anti-PD-1 and/or anti-PD-L1 antibody and a tumor proportion score has been determined prior to treatment with the anti-PD-1 and/or anti-PD-L1 antibody. In some embodiments, a subject or patient with refractory or resistant NSCLC has been previously treated with an anti-PD-L1 antibody and a tumor proportion score has been determined prior to the anti-PD-L1 antibody treatment.

In some embodiments, NSCLC is NSCLC exhibiting a Tumor Proportion Score (TPS), or the percentage of viable tumor cells obtained from a patient prior to anti-PD-1 or anti-PD-L1 therapy, the percentage of PD-L1 protein exhibiting partial or complete membrane staining at any intensity is less than 1% (TPS < 1%). In some embodiments, NSCLC is NSCLC：<50％、<45％、<40％、<35％、<30％、<25％、<20％、<15％、<10％、<9％、<8％、<7％、<6％、<5％、<4％、<3％、<2％、<1％、<0.9％、<0.8％、<0.7％、<0.6％、<0.5％、<0.4％、<0.3％、<0.2％、<0.1％、<0.09％、<0.08％、<0.07％、<0.06％、<0.05％、<0.04％、<0.03％、<0.02％ and <0.01% exhibiting a TPS selected from the group consisting of. In some embodiments, the NSCLC is a NSCLC that exhibits a TPS selected from: about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, and about 0.01%. In some embodiments, the NSCLC is NSCLC exhibiting TPS between 0% and 1%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.9%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.8%. In some embodiments, the NSCLC is NSCLC exhibiting TPS between 0% and 0.7%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.6%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.5%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.4%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.3%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.2%. In some embodiments, the NSCLC is NSCLC that exhibits TPS between 0% and 0.1%. TPS can be measured by methods known in the art, such as those described in Hirsch et al, journal of thoracic oncology (J. Thorac. Oncol.) 2017,12,208-222 or those used to determine TPS prior to treatment with palbocuzumab (pembrolizumab) or other anti-PD-1 or anti-PD-L1 therapies. Methods approved by the U.S. food and drug administration for measuring TPS may also be used. In some embodiments, PD-L1 is exosome PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

In some embodiments, partial membrane staining comprises 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more. In some embodiments, the complete membrane staining comprises about 100% membrane staining.

In some embodiments, testing PD-L1 may involve determining the level of PD-L1 in the patient's serum. In these embodiments, measurement of PD-L1 in patient serum removes uncertainty of tumor heterogeneity and discomfort of patient with continuous biopsy.

In some embodiments, an elevated level of soluble PD-L1 as compared to baseline or standard is correlated with a prognosis of exacerbation of NSCLC. See, e.g., okuma et al, clinical lung cancer (Clinical Lung Cancer), 2018,19,410-417; VECCHIARELLI et al, tumor target (Oncotarget), 2018,9,17554-17563. In some embodiments, PD-L1 is exosome PD-L1. In some embodiments, PD-L1 is expressed on circulating tumor cells.

In some embodiments, the invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a population of tumor-infiltrating lymphocytes (TILs) to a subject or patient in need thereof, wherein the subject or patient has at least one of:

A predetermined Tumor Proportion Score (TPS) of pd-L1 <1%,

TPS fraction of PD-L1 is 1% -49%, or

One or more predetermined deletions of the driving mutations,

Wherein the driving mutation is selected from the group consisting of: EGFR mutation, EGFR insertion, EGFR exon 20 mutation, KRAS mutation, BRAF mutation, ALK mutation, C-ROS mutation (ROS 1 mutation), ROS1 fusion, RET mutation, RET fusion, ERBB2 mutation, ERBB2 amplification, BRCA mutation, MAP2K1 mutation, PIK3CA, CDKN2A, PTEN mutation, UMD mutation, NRAS mutation, KRAS mutation, NF1 mutation, MET splice and/or altered MET signal, TP53 mutation, crebp mutation, KMT2C mutation, KMT2D mutation, ARID1A mutation, RB1 mutation, ATM mutation, SETD2 mutation, FLT3 mutation, PTPN11 mutation, FGFR1 mutation, EP300 mutation, MYC mutation, EZH2 mutation, JAK2 mutation, FBXW7 mutation, CCND3 mutation and GNA11 mutation, wherein the method comprises:

(a) Obtaining and/or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) Adding a first TIL population to a closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2, producing a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area, the first amplification being performed for about 3 to 14 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(d) Performing a second amplification by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), producing a third TIL population, wherein the second amplification is performed for about 7 days to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(g) Cryopreserving the infusion bag from step (f) containing the collected TIL population by a cryopreservation process; and

(H) Administering to the subject or patient a therapeutically effective dose of a third TIL population from the infusion bag in step (g).

In some embodiments, the invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a population of tumor-infiltrating lymphocytes (TILs) to a patient in need thereof, wherein the method comprises:

(a) The patient's tumor was tested for PD-L1 expression and Tumor Proportion Score (TPS) for PD-L1,

(B) Testing the patient for the absence of more than one driving mutation, wherein the driving mutation is selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20 mutations, KRAS mutations, BRAF mutations, ALK mutations, C-ROS mutations (ROS 1 mutations), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET splicing and/or altered MET signaling, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations and GNA11 mutations,

(C) Determining that the patient has a PD-L1 TPS score of about 1% to about 49% and determining that the patient also has no driving mutations,

(D) Obtaining and/or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(e) Adding a first TIL population to a closed system;

(f) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2, producing a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area, the first amplification being performed for about 3 to 14 days to obtain the second population of TILs, the transition from step (e) to step (f) being performed without opening the system;

(g) Performing a second amplification by supplementing a cell culture medium of a second population of TILs with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a third population of TILs, wherein the second amplification is performed for about 7 to 14 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (f) to step (g) being performed without opening the system;

(h) Collecting the population of therapeutic TILs obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system;

(i) Transferring the collected TIL population from step (e) to an infusion bag, wherein the transferring of steps (e) to (f) is performed without opening the system;

(j) Cryopreserving the infusion bag from step (f) containing the collected TIL population by a cryopreservation process; and

(K) Administering to the subject or patient a therapeutically effective dose of a third population of TILs from the infusion bag in step (g).

(C) Determining that the patient has a PD-L1 TPS score of less than about 1% and determining that the patient also has no driving mutations,

(D) Obtaining and/or receiving a first TIL population from a tumor resected from the subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(e) Adding a first TIL population to a closed system;

(f) Performing a first amplification by culturing the first population of TILs in a cell culture medium comprising IL-2, producing a second population of TILs, wherein the first amplification is performed in a closed vessel providing a first gas permeable surface area, the first amplification being performed for about 3 to 14 days to obtain the second population of TILs, the transition from step (e) to step (f) being performed without opening the system;

(K) Administering to the subject or patient a therapeutically effective dose of the third TIL population from the infusion bag in step (g).

(B) Testing the patient for the absence of more than one driving mutation, wherein the driving mutation is selected from the group consisting of: EGFR mutation, EGFR insertion, KRAS mutation, BRAF mutation, ALK mutation, c-ROS mutation (ROS 1 mutation), ROS1 fusion, RET mutation or RET fusion,

(e) Adding a first TIL population to a closed system;

(g) Performing a second amplification by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a third TIL population, wherein the second amplification is performed for about 7 days to 14 days to obtain a third TIL population, the third TIL population being the therapeutic TIL population, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (f) to step (g) being performed without opening the system;

In other embodiments, the invention provides a method for treating a subject having cancer comprising administering to the subject a therapeutically effective dose of a therapeutic TIL population described herein.

In other embodiments, the invention provides a method for treating a subject having cancer comprising administering to the subject a therapeutically effective dose of a TIL composition described herein.

In other embodiments, the invention provides modified methods for treating a subject having a cancer described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to the separate administration of a therapeutically effective dose of the therapeutic TIL populations and TIL compositions described herein.

In other embodiments, the invention provides a modified method for treating a subject having a cancer as described herein, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for five days.

In other embodiments, the invention provides a modified method for treating a subject having a cancer described herein, the method further comprising the step of beginning the treatment of the subject with a high dose IL-2 regimen the next day after administration of TIL cells to the subject.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg in the form of a 15 minute bolus intravenous infusion every eight hours until tolerized.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is a solid tumor.

In other embodiments, the invention provides modified methods for treating a subject having a cancer described herein, wherein the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), neuroblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

In other embodiments, the invention provides modified methods for treating a subject having a cancer described herein, wherein the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is melanoma.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is HNSCC.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is cervical cancer.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is NSCLC.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is neuroglioblastoma (including GBM).

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is a high mutation cancer.

In other embodiments, the invention provides modified methods for treating a subject having a cancer as described herein, wherein the cancer is a pediatric hypermutated cancer.

In other embodiments, the invention provides a therapeutic TIL population described herein in a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

In other embodiments, the invention provides a TIL composition described herein for use in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In other embodiments, the invention provides a modified therapeutic TIL population described herein or a TIL composition described herein, wherein a non-myeloablative lymphocyte depletion regimen has been administered to a subject prior to administration of a therapeutically effective dose of a therapeutic TIL population described herein or a TIL composition described herein to the subject.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for five days.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, further comprising the step of starting the treatment of the patient with the high dose IL-2 regimen the day after the administration of the TIL cells to the patient.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg in the form of a 15 minute bolus intravenous infusion every eight hours until tolerized.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is a solid tumor.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is HNSCC.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is cervical cancer.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is NSCLC.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is neuroglioblastoma.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is a hypermutated cancer.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is pediatric hypermutated cancer.

In other embodiments, the invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective dose of a therapeutic TIL population.

In other embodiments, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of a TIL composition described in any of the preceding paragraphs.

In other embodiments, the invention provides the use of a therapeutic TIL population described herein or a TIL composition described herein in a method of treating cancer in a patient, the method comprising administering to the patient a non-myeloablative lymphocyte depletion regimen, followed by administering to the subject a therapeutically effective dose of a therapeutic TIL population described in any of the preceding paragraphs or a therapeutically effective dose of a TIL composition described herein.

1. Combination with PD-1 and PD-L1 inhibitors

In some embodiments, the TIL therapy provided to the cancer patient may comprise treatment with a therapeutic TIL population alone, or may comprise combination therapy comprising TIL and one or more PD-1 and/or PD-L1 inhibitors.

Programmed death 1 (PD-1) is a 288 amino acid transmembrane immune checkpoint receptor protein expressed by T cells, B cells, natural Killer (NK) T cells, activated monocytes and dendritic cells. PD-1, also known as CD279, belongs to the CD28 family and is encoded in humans by the PDcd gene on chromosome 2. PD-1 consists of an immunoglobulin (Ig) superfamily domain, a transmembrane domain, and an intracellular domain containing an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) and an Immunoreceptor Tyrosine Switching Motif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play an important role in immune tolerance as described in Keir et al, immunology annual comment 2008,26,677-704. PD-1 provides an inhibitory signal that negatively regulates T cell immune responses. PD-L1 (also known as B7-H1 or CD 274) and PD-L2 (also known as B7-DC or CD 273) are expressed on tumor cells and stromal cells, which may encounter activated T cells expressing PD-1, resulting in immunosuppression of the T cells. PD-L1 is a 290 amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9. Blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2 using PD-1 inhibitors, PD-L1 inhibitors, and/or PD-L2 inhibitors may overcome immune resistance as shown by recent clinical studies, for example, topalian et al, new england journal of medicine (n.eng.j.med.) 2012,366,2443-54. PD-L1 is expressed on many tumor cell lines, while PD-L2 is expressed predominantly on dendritic cells and some tumor strains. In addition to T cells (which inducible express PD-1 upon activation), PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes and dendritic cells.

In some embodiments, the TIL and PD-1 inhibitor are administered as a combination therapy or co-therapy for treating NSCLC.

In some embodiments, NSCLC has not undergone prior therapy. In some embodiments, the PD-1 inhibitor is administered as a first line therapy or initial therapy. In some embodiments, the PD-1 inhibitor is administered as a first line therapy or initial therapy in combination with a TIL as described herein.

In some embodiments, the PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail in the following paragraphs. With respect to PD-1 inhibitors, the terms "inhibitor," "antagonist," and "blocker" are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-1 inhibitor as an antibody may refer to a compound or antigen-binding fragment, variant, conjugate or biological analogue thereof. For the avoidance of doubt, references herein to a PD-1 inhibitor may also refer to a small molecule compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

In some embodiments, the PD-1 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof, including Fab fragments thereof, or single chain variable fragments (scFv). In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In some embodiments, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competes for binding to PD-1, and/or binds to an epitope on PD-1. In some embodiments, the antibody competes for binding to PD-1, and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds to human PD-1 with a KD of about 100pM or less, binds to human PD-1 with a KD of about 90pM or less, binds to human PD-1 with a KD of about 80pM or less, binds to human PD-1 with a KD of about 70pM or less, binds to human PD-1 with a KD of about 60pM or less, binds to human PD-1 with a KD of about 50pM or less, binds to human PD-1 with a KD of about 40pM or less, binds to human PD-1 with a KD of about 30pM or less, binds to human PD-1 with a KD of about 20pM or less, binds to human PD-1 with a KD of about 10pM or less, or binds to human PD-1 with a KD of about 1pM or less.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds human PD-1 at k _assoc of about 7.5x10 ⁵ l/m·s or faster, binds human PD-1 at k _assoc of about 7.5x10 ⁵ 1/m·s or faster, binds human PD-1 at k _assoc of about 8 x 10 ⁵ 1/m·s or faster, binds human PD-1 at k _assoc of about 8.5x10 ⁵/m·s or faster, binds human PD-1 at k _assoc of about 9 x 10 ⁵ 1/m·s or faster, binds human PD-1 at k _assoc of about 9.5x10 ⁵ l/m·s or faster, or binds human PD-1 at k _assoc of about 1 x 10 ⁶ l/m·s or faster.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds human PD-1 at a k _dissoc of about 2x 10 ^-5/s or slower, binds human PD-1 at a k _dissoc of about 2.1 x 10 ^-5/s or slower, binds human PD-1 at a k _dissoc of about 2.2 x 10 ^-5 1/s or slower, binds human PD-1 at a k _dissoc of about 2.3 x 10 ^-5/s or slower, binds human PD-1 at a k _dissoc of about 2.4 x 10 ^-5/s or slower, binds human PD-1 at a k _dissoc of about 2.5 x 10 ^-5/s or slower, binds human PD-1 at a k _dissoc of about 2.6 x 10 ^-5/s or slower, binds human PD-1 at a k _dissoc of about 2.7 x 10 ^-5/s or slower, binds human PD-1 at a k _dissoc of about 2.8 x 10/s or slower, binds human PD-1 at a k _dissoc of about 2.5 x 10 x 36/s or slower, binds human PD-1 at a k _dissoc of about 2.5 x 10 x 36/s or slower, binds human PD-1 at a k 3735 of about 2.6 x 10 x 36/s or slower, or binds human PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 9nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 8nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 7nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 6nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-L2 with an IC50 of about 4nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-L1 with an IC50 of about 3nM or less, blocks or inhibits the binding of human PD-L2 to human PD-L1 or human PD-L2 with an IC-1 with an IC50 of about 6nM or less than about 6 nM.

In some embodiments, the PD-1 inhibitor is nivolumab (commercially available from the company bezels, magnaporthe, OPDIVO) or a biological analog, antigen-binding fragment, conjugate, or variant thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-1 receptor. In some embodiments, the anti-PD-1 antibody is an immunoglobulin G4 kappa anti (human CD 274) antibody. Nawuzumab was assigned Chemical Abstracts (CAS) accession numbers 946414-94-4 and also known as 5C4, BMS-936558, MDX-1106, and ONO-4538. The preparation and characterization of nivolumab is described in U.S. patent No. 8,008,449 and international patent publication No. WO 2006/121168, the disclosures of which are incorporated herein by reference. Clinical safety and efficacy of nivolumab in various forms of Cancer has been described in Wang et al, cancer immunology study (Cancer Immunol res.) 2014,2,846-56; page et al, medical annual assessment (Ann. Rev. Med.), 2014,65,185-202; and Weber et al, journal of clinical oncology, 2013,31,4311-4318, the disclosures of which are incorporated herein by reference. The amino acid sequence of nivolumab is set forth in table 26. Nivolumab has intra-heavy chain disulfide bonds at 22-96, 140-196, 254-314, 360-418, 22"-96", 140"-196", 254"-314", and 360 "-418"; having a light chain internal disulfide bond at 23'-88', 134'-194', 23 '"-88'" and 134 '"-194'"; having a heavy chain-light chain inter-disulfide bond at 127-214', 127 "-214'"; having a heavy chain-heavy chain interchain disulfide bond at 219-219 "and 222-222"; and has an N-glycosylation site (H CH 284.4) at 290, 290'.

In some embodiments, the PD-1 inhibitor comprises SEQ ID NO:158 and the heavy chain shown in SEQ ID NO: 159. In some embodiments, the PD-1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO:158 and SEQ ID NO:159, or an antigen binding fragment, fab fragment, single chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 99% identity. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 98% identity. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 97% identity. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 96% identity. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 95% identity.

In some embodiments, the PD-1 inhibitor comprises the heavy and light chain CDRs or Variable Regions (VRs) of nivolumab. In some embodiments, the PD-1 inhibitor heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:160, and the PD-1 inhibitor light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:161 and conservative amino acid substitutions thereof. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:160 and SEQ ID NO:161 has at least 99% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:160 and SEQ ID NO:161 has at least 98% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:160 and SEQ ID NO:161 has at least 97% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:160 and SEQ ID NO:161 has at least 96% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:160 and SEQ ID NO:161 has at least 95% identity to the V _H region and the V _L region.

In some embodiments, the PD-1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO: 162. SEQ ID NO:163 and SEQ ID NO:164 and the heavy chain CDR1, CDR2 and CDR3 domains thereof with conservative amino acid substitutions; and having the amino acid sequence of SEQ ID NO: 165. SEQ ID NO:166 and SEQ ID NO:167 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof. In some embodiments, the antibody competes for binding to and/or to: the same epitope on PD-1 as any of the antibodies previously described.

In some embodiments, the PD-1 inhibitor is an anti-PD-1 biological analog monoclonal antibody approved by a drug administration reference to nivolumab. In some embodiments, a biological analog comprises an anti-PD-1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. anti-PD-1 antibodies may be available to drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients included in a reference drug or reference biologic, wherein the reference drug or reference biologic is nivolumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients included in a reference drug or reference biologic, wherein the reference drug or reference biologic is nivolumab.

Table 26: reference is made to the amino acid sequence of the PD-1 inhibitor of nivolumab.

In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, and the nivolumab is administered at a dose of about 0.5mg/kg to about 10mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, the nivolumab being administered at the following doses: about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about 4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.5mg/kg, about 7mg/kg, about 7.5mg/kg, about 8mg/kg, about 8.5mg/kg, about 9mg/kg, about 9.5mg/kg or about 10mg/kg. In some embodiments, administration of nivolumab begins 1, 2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1, 2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, and the nivolumab is administered at a dose of about 200mg to about 500mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, the nivolumab being administered at the following doses: about 200mg, about 220mg, about 240mg, about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg or about 500mg. In some embodiments, administration of nivolumab begins 1,2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, and the nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, administration of nivolumab begins 1,2,3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma. In some embodiments, the nivolumab is administered to treat unresectable or metastatic melanoma at about 240mg every 2 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma, and is administered at about 480mg every 4 weeks. In some embodiments, the nivolumab is administered to treat unresectable or metastatic melanoma at about 1mg/kg of nivolumab per 3 weeks on the same day, followed by 3mg/kg of ipilimumab for 4 doses, followed by 240mg per 2 weeks or 480mg per 4 weeks.

In some embodiments, nivolumab is administered to assist in the treatment of melanoma. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to aid in the treatment of melanoma. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to aid in the treatment of melanoma. In some embodiments, administration of nivolumab begins 1, 2,3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1, 2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1, 2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered at about 3mg/kg every 2 weeks and ipilimumab is administered at about 1mg/kg every 6 weeks to treat metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered at about 360mg every 3 weeks, plus 1mg/kg of ipilimumab every 6 weeks with 2 cycles of platinum-containing dual chemotherapy to treat metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered at about 240mg every 2 weeks or 480mg every 4 weeks to treat metastatic non-small cell lung cancer. In some embodiments, administration of nivolumab begins 1,2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat small cell lung cancer. In some embodiments, the nivolumab is administered at about 240mg every 2 weeks to treat small cell lung cancer. In some embodiments, administration of nivolumab begins 1, 2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1, 2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered at about 360mg every 3 weeks and ipilimumab at 1mg/kg every 6 weeks to treat malignant pleural mesothelioma. In some embodiments, administration of nivolumab begins 1, 2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1, 2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 3mg/kg, then ipilimumab is administered at about 1mg/kg for 4 doses every 3 weeks on the same day, followed by 240mg of nivolumab every 2 weeks to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 3mg/kg, then ipilimumab is administered at about 1mg/kg for 4 doses on the same day every 3 weeks, followed by 240mg every 2 weeks and 480mg of nivolumab every 4 weeks to treat advanced renal cell carcinoma. In some embodiments, administration of nivolumab begins 1,2, 3,4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2, 3,4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat a typical hodgkin's lymphoma. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat a typical hodgkin's lymphoma. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat a typical hodgkin's lymphoma. In some embodiments, administration of nivolumab begins 1, 2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1, 2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat recurrent or metastatic head and neck squamous cell carcinoma. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat recurrent or metastatic head and neck squamous cell carcinoma. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat recurrent or metastatic head and neck squamous cell carcinoma. In some embodiments, administration of nivolumab begins 1,2,3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat locally advanced or metastatic urothelial cancer. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat locally advanced or metastatic urothelial cancer. In some embodiments, administration of nivolumab begins 1,2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer. In some embodiments, nivolumab is administered to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult and pediatric patients. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult and pediatric patients of > 40 kg. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult and pediatric patients of > 40 kg. In some embodiments, administration of nivolumab begins 1,2,3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered at about 3mg/kg every 2 weeks to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in pediatric patients of <40 kg. In some embodiments, nivolumab is administered at about 3mg/kg, followed by 1mg/kg of ipilimumab on the same day every 3 weeks for 4 doses, followed by 240mg of nivolumab every 2 weeks to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult and pediatric patients of > 40 kg. In some embodiments, nivolumab is administered at about 3mg/kg, followed by 1mg/kg of ipilimumab on the same day every 3 weeks for 4 doses, followed by 480mg of nivolumab every 4 weeks to treat high microsatellite instability (MSI-H) or mismatch repair defect (dMMR) metastatic colorectal cancer in adult and pediatric patients of > 40 kg. In some embodiments, administration of nivolumab begins 1,2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1,2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 1mg/kg, then 3mg/kg of ipilimumab is administered on the same day for 4 doses every 3 weeks, followed by 240mg of nivolumab every 2 weeks to treat hepatocellular carcinoma. In some embodiments, the nivolumab is administered at about 1mg/kg, then 3mg/kg of ipilimumab is administered on the same day for 4 doses every 3 weeks, followed by 480mg of nivolumab every 4 weeks to treat hepatocellular carcinoma. In some embodiments, administration of nivolumab begins 1, 2, 3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1, 2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, nivolumab is administered to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat esophageal squamous cell carcinoma. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat esophageal squamous cell carcinoma. In some embodiments, administration of nivolumab begins 1, 2,3, 4, or 5 days after administration of IL-2. In some embodiments, administration of nivolumab begins 1, 2, or 3 days after administration of IL-2. In some embodiments, nivolumab may also be administered 1, 2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the PD-1 inhibitor comprises palbociclib (commercially available as KEYTRUDA from merck corporation of chenille, new jersey) or an antigen binding fragment, conjugate, or variant. Palbociclib was assigned CAS accession number 1374853-91-4 and is also known as lanlizumab, MK-3475, and SCH-900475. Palbociclib has an immunoglobulin G4 anti-human protein PDCD1 (programmed cell death 1)) antibody containing (human mouse monoclonal heavy chain) disulfide bond and human mouse monoclonal light chain dimer structure. The structure of palbociclib can also be described as an immunoglobulin G4 anti (human programmed cell death 1) antibody; contains disulfide bonds of a humanized mouse monoclonal [ 228-L-proline (H10-S > P) ] gamma 4 heavy chain (134-218 ') and disulfide bonds of a humanized mouse monoclonal kappa light chain dimer (226-226': 229-229 "). Characteristics, uses and preparation of palbociclib are described in international patent publication No. WO 2008/156712 A1, U.S. patent No. 8,354,509, and U.S. patent application publication nos. US2010/0266617 A1, US2013/0108651 A1 and US 2013/0109843A2, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of palbociclib in various forms of cancer is described in Fuerst, oncology Times, 2014,36,35-36; robert et al, lancet, 2014,384,1109-17; and Thomas et al, 2014,14,1061-1064, biological therapeutic expert opinion (exp. Opin. Biol. Ther.). The amino acid sequence of palbociclib is set forth in table 27. The palbociclib comprises the following disulfide bonds ：22-96、22"-96"、23'-92'、23"'-92"'、134-218'、134"-218"'、138'-198'、138"'-198"'、147-203、147"-203"、226-226"、229-229"、261-321、261"-321"、367-425 and 367"-425"; the following glycosylation sites (N): asn-297 and Asn-297). Palboc Li Zhushan antibody is an IgG4/κ isotype with a stabilizing S228P mutation in the Fc region; insertion of this mutation in the IgG4 hinge region prevents the formation of half the molecule normally observed for IgG4 antibodies. Palbociclib is heterogeneously glycosylated at Asn297 within the Fc domain of each heavy chain, such that the molecular weight of the intact antibody is about 149kDa. The primary glycoform of palbociclib is the fucosylated degalactosylation ditolysaccharide form (G0F).

In some embodiments, the PD-1 inhibitor comprises SEQ ID NO:168 and the heavy chain shown in SEQ ID NO: 169. In some embodiments, the PD-1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO:168 and SEQ ID NO:169, or antigen binding fragments, fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:168 and SEQ ID NO:169 has at least 99% identity to the heavy and light chains. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:168 and SEQ ID NO:169 has at least 98% identity between the heavy and light chains. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:168 and SEQ ID NO:169 has at least 97% identity to the heavy and light chains. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:168 and SEQ ID NO:169 has at least 96% identity. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:168 and SEQ ID NO:169 has at least 95% identity to the heavy and light chains.

In some embodiments, the PD-1 inhibitor comprises the heavy and light chain CDRs or Variable Regions (VRs) of palbociclizumab. In some embodiments, the PD-1 inhibitor heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:170, and the PD-1 inhibitor light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:171 and conservative amino acid substitutions thereof. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:170 and SEQ ID NO:171 has at least 99% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:170 and SEQ ID NO:171 has at least 98% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:170 and SEQ ID NO:171 has at least 97% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:170 and SEQ ID NO:171 has at least 96% identity to the V _H region and the V _L region. In some embodiments, the PD-1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:170 and SEQ ID NO:171 has at least 95% identity to the V _H region and the V _L region.

In some embodiments, the PD-1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO: 172. SEQ ID NO:173 and SEQ ID NO:174 and the heavy chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof; and having the amino acid sequence of SEQ ID NO: 175. SEQ ID NO:176 and SEQ ID NO:177 and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains. In some embodiments, the antibody competes for binding to and/or to: the same epitope on PD-1 as any of the antibodies previously described.

In some embodiments, the PD-1 inhibitor is an anti-PD-1 biological analog monoclonal antibody approved by a drug administration reference palbociclib. In some embodiments, a biological analog comprises an anti-PD-1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is pam Li Zhushan antibody. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized anti-PD-1 antibody, wherein the anti-PD-1 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is pamphlet Li Zhushan antibody. anti-PD-1 antibodies may be available to drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is pamphlet-Li Zhushan antibody. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is pamphlet-Li Zhushan antibody.

Table 27: reference to the amino acid sequence of a PD-1 inhibitor of palbociclizumab

In some embodiments, the PD-1 inhibitor is palbociclib or a biological analog thereof, and the palbociclib Li Zhushan is administered at a dose of about 0.5mg/kg to about 10mg/kg. In some embodiments, the PD-1 inhibitor is palbociclizumab or a biological analog thereof, and the palbociclib Li Zhushan is administered at the following doses: about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about 4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.5mg/kg, about 7mg/kg, about 7.5mg/kg, about 8mg/kg, about 8.5mg/kg, about 9mg/kg, about 9.5mg/kg or about 10mg/kg. In some embodiments, palbociclib administration is initiated 1,2,3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the PD-1 inhibitor is palbociclib or a biological analog thereof, wherein the palbociclib Li Zhushan is administered at a dose of about 200mg to about 500mg. In some embodiments, the PD-1 inhibitor is palbociclib or a biological analog thereof, and the nivolumab is administered at the following doses: about 200mg, about 220mg, about 240mg, about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg or about 500mg. In some embodiments, palbociclib administration is initiated 1,2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the PD-1 inhibitor is palbociclib or a biological analog thereof, wherein the palbociclib is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, palbociclib administration is initiated 1, 2,3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered to treat melanoma. In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat melanoma. In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat melanoma. In some embodiments, palbociclib administration is initiated 1,2, 3,4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3,4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered to treat NSCLC. In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat NSCLC. In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat NSCLC. In some embodiments, palbociclib administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered to treat Small Cell Lung Cancer (SCLC). In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat SCLC. In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat SCLC. In some embodiments, palbociclib administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan is administered to treat Head and Neck Squamous Cell Carcinoma (HNSCC). In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat HNSCC. In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat HNSCC. In some embodiments, palbociclib administration is initiated 1,2,3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat a typical hodgkin's lymphoma (cHL) or primary mediastinum large B cell lymphoma (PMBCL). In some embodiments, the adult human is administered pamphlet Li Zhushan antibody at about 400mg every 6 weeks to treat a typical hodgkin's lymphoma (cHL) or primary mediastinum large B-cell lymphoma (PMBCL). In some embodiments, the pediatric administration of the pamphlet Li Zhushan antibody every 3 weeks at about 2mg/kg (up to 200 mg) treats a typical hodgkin's lymphoma (cHL) or primary mediastinum large B cell lymphoma (PMBCL). In some embodiments, palbociclib administration is initiated 1,2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat urothelial cancer. In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat urothelial cancer. In some embodiments, palbociclib administration is initiated 1, 2, 3,4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3,4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) cancer. In some embodiments, the adult human is administered pamphlet Li Zhushan antibody at about 400mg every 6 weeks to treat MSI-H or dMMR cancer. In some embodiments, the pediatric administration of the pamphlet Li Zhushan antibody to treat MSI-H or dMMR cancer is about 2mg/kg (up to 200 mg) every 3 weeks. In some embodiments, palbociclib administration is initiated 1,2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, palbociclib administration is initiated 1,2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat high microsatellite instability (MSI-H) or mismatch repair deficient colorectal cancer (dMMR CRC). In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat MSI-H or dMMR CRC. In some embodiments, palbociclib administration is initiated 1, 2,3,4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2,3,4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat gastric cancer. In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat gastric cancer. In some embodiments, palbociclib administration is initiated 1,2,3,4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2,3,4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat esophageal cancer. In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat esophageal cancer. In some embodiments, palbociclib administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat cervical cancer. In some embodiments, the pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat cervical cancer. In some embodiments, palbociclib administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat hepatocellular carcinoma (HCC). In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat HCC. In some embodiments, palbociclib administration is initiated 1,2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the adult human is administered pamphlet Li Zhushan antibody at about 200mg every 3 weeks to treat Merck Cell Carcinoma (MCC). In some embodiments, the adult human administers the pamphlet Li Zhushan antibody at about 400mg every 6 weeks to treat MCC. In some embodiments, the pediatric administration of pamoic Li Zhushan antibody to treat MCC every 3 weeks is at about 2mg/kg (up to 200 mg). In some embodiments, palbociclib administration is initiated 1,2,3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, palbociclib administration is initiated 1,2,3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat Renal Cell Carcinoma (RCC). In some embodiments, pamglizumab is administered at about 400mg every 6 weeks and axitinib (axitinib mg) is orally administered twice daily to treat RCC. In some embodiments, palbociclib administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat endometrial cancer. In some embodiments, palbociclib is administered at about 400mg every 6 weeks and lenvatinib (lenvatinib) for non-MSI-H or dMMR tumors is orally administered 20mg once a day to treat endometrial cancer. In some embodiments, palbociclib administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the pamphlet Li Zhushan antibody is administered every 3 weeks at about 200mg to treat high tumor mutational burden (TMB-H) cancer in an adult. In some embodiments, the adult is administered pamphlet Li Zhushan antibody at about 400mg every 6 weeks to treat TMB-H cancer. In some embodiments, the pediatric administration of the pamphlet Li Zhushan antibody to treat TMB-H cancer is about 2mg/kg (up to 200 mg) every 3 weeks. In some embodiments, palbociclib administration is initiated 1,2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat skin squamous cell carcinoma (cSCC). In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat cSCC. In some embodiments, palbociclib administration is initiated 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1, 2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, pamphlet Li Zhushan antibody is administered at about 200mg every 3 weeks to treat Triple Negative Breast Cancer (TNBC). In some embodiments, pamphlet Li Zhushan antibody is administered at about 400mg every 6 weeks to treat TNBC. In some embodiments, palbociclib administration is initiated 1,2, 3, 4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, if the patient or subject is an adult, i.e., for treatment of an adult indication, an additional 400mg dosing regimen every 6 weeks may be employed. In some embodiments, palbociclib administration is initiated 1,2, 3,4, or 5 days after IL-2 administration. In some embodiments, palbociclib administration is initiated 1,2, or 3 days after IL-2 administration. In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, 3,4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, pamphlet Li Zhushan antibody may also be administered 1,2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 clone J43 (catalog No. BE 0033-2) and RMP1-14 (catalog No. BE 0146) (Bio X Cell, inc., of new hampson Xia Zhou parisons, usa). A variety of commercially available anti-PD-1 antibodies are known to those of ordinary skill in the art.

In some embodiments, the PD-1 inhibitor is an antibody disclosed in U.S. patent No. 8,354,509 or U.S. patent application publication nos. 2010/0266617A1, 2013/0108651A1, 2013/0109843A2, the disclosures of which are incorporated herein by reference. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody described in U.S. patent nos. 8,287,856, 8,580,247, and 8,168,757, and U.S. patent application publication nos. 2009/0028857A1, 2010/0285013A1, 2013/0022600A1, and 2011/0008369A1, the teachings of which are incorporated herein by reference. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in U.S. patent No. 8,735,553B1, the disclosure of which is incorporated herein by reference. In some embodiments, the PD-1 inhibitor is Pi Lizhu mab, also known as CT-011, described in U.S. patent No. 8,686,119, the disclosure of which is incorporated herein by reference.

In some embodiments, the PD-1 inhibitor may be a small molecule or peptide derivative, such as described in U.S. patent nos. 8,907,053, 9,096,642, and 9,044,442, and U.S. patent application publication No. US 2015/0087581; 1,2, 4-oxadiazole compounds and derivatives, such as 1,2, 4-oxadiazole compounds and derivatives described in U.S. patent application publication 2015/0074350; cyclic peptide mimetic compounds and derivatives, such as those described in U.S. patent application publication No. US 2015/0074402; cyclic compounds and derivatives, such as those described in U.S. patent application publication No. US 2015/0125091; 1,3, 4-oxadiazole and 1,3, 4-thiadiazole compounds and derivatives, such as 1,3, 4-oxadiazole and 1,3, 4-thiadiazole compounds and derivatives described in International patent application publication No. WO 2015/033301; peptide-based compounds and derivatives, such as those described in International patent application publication Nos. WO 2015/036927 and WO 2015/04490; or peptide-based macrocyclic compounds and derivatives, such as described in U.S. patent application publication No. US 2014/0294898; their respective disclosures are incorporated herein by reference in their entirety. In some embodiments, the PD-1 inhibitor is a cimeprol Li Shan antibody (cemiplimab), which is commercially available from regenerator corporation (Regeneron, inc.).

In some embodiments, the TIL and PD-L1 inhibitor or PD-L2 inhibitor are administered as a combination therapy or co-therapy for treating NSCLC.

In some embodiments, NSCLC has not undergone prior therapy. In some embodiments, the PD-L1 inhibitor or PD-L2 inhibitor is administered as a first line therapy or initial therapy. In some embodiments, the PD-L1 inhibitor or PD-L2 inhibitor is administered as a first line therapy or initial therapy in combination with TIL as described herein.

In some embodiments, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors, antagonists or blockers described in more detail in the following paragraphs. With respect to PD-L1 and PD-L2 inhibitors, the terms "inhibitor," "antagonist," and "blocker" are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor as an antibody may refer to a compound or antigen-binding fragment, variant, conjugate or biological analogue thereof. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor may also refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

In some embodiments, the compositions, processes, and methods described herein comprise PD-L1 or PD-L2 inhibitors. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof, including Fab fragments or single chain variable fragments (scFv) thereof. In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competes for binding to PD-L1 or PD-L2 and/or to an epitope on PD-L1 or PD-L2. In some embodiments, the antibody competes for binding to PD-L1 or PD-L2, and/or binds to an epitope on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selective for PD-L1 because the concentration of the compound that binds or interacts with PD-L1 is much lower than the concentration of the compound that binds or interacts with other receptors, including the PD-L2 receptor. In certain embodiments, the binding constant of a compound to a PD-L1 receptor is at least about 2-fold higher, about 3-fold higher, about 5-fold higher, about 10-fold higher, about 20-fold higher, about 30-fold higher, about 50-fold higher, about 100-fold higher, about 200-fold higher, about 300-fold higher, or about 500-fold higher than the binding constant of the PD-L2 receptor.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2 because the concentration of the compound that binds or interacts with PD-L2 is much lower than the concentration of the compound that binds or interacts with other receptors, including the PD-L1 receptor. In certain embodiments, the binding constant of a compound to a PD-L2 receptor is at least about 2-fold higher, about 3-fold higher, about 5-fold higher, about 10-fold higher, about 20-fold higher, about 30-fold higher, about 50-fold higher, about 100-fold higher, about 200-fold higher, about 300-fold higher, or about 500-fold higher than the binding constant of the PD-L1 receptor.

Without being bound by any theory, it is believed that tumor cells express PD-L1 and T cells express PD-1. However, the expression of PD-L1 by tumor cells is not required for the efficacy of PD-1 or PD-L1 inhibitors or blockers. In some embodiments, the tumor cell expresses PD-L1. In other embodiments, the tumor cells do not express PD-L1. In some embodiments, the methods may include a combination of PD-1 and PD-L1 antibodies (e.g., PD-1 and PD-L1 antibodies described herein) with TIL. The combination of PD-1 and PD-L1 antibodies with TIL may be administered simultaneously or sequentially.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that binds to human PD-L1 and/or PD-L2 with a KD of about 100pM or less, binds to human PD-L1 and/or PD-L2 with a KD of about 90pM or less, binds to human PD-L1 and/or PD-L2 with a KD of about 80pM or less, binds to human PD-L1 and/or PD-L2 with a KD of about 70pM or less, binds to human PD-L1 and/or PD-L2 with a KD of about 60pM or less, binds to human PD-L1 and/or PD-L2 with a KD of about 50pM or less, binds to human PD-L1 and/or PD-L2 with a KD of about 40pM or less, or binds to human PD-L1 and/or PD-L2 with a KD of about 30pM or less.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that binds human PD-L1 and/or PD-L2 at a k _assoc of about 7.5x10 ⁵ 1/m·s or faster, binds human PD-L1 and/or PD-L2 at a k _assoc of about 8 x10 ⁵/m·s or faster, binds human PD-L1 and/or PD-L2 at a k _assoc of about 8.5x10 ⁵/m·s or faster, binds human PD-L1 and/or PD-L2 at a k _assoc of about 9 x10 ⁵ 1/m·s or faster, binds human PD-L1 and/or PD-L2 at a k _assoc of about 9.5x10 ⁵/m·s or faster, or binds human PD-L1 and/or PD-L2 at a k ⁶/m·s or faster, or binds human PD-L1 and/or PD-L2 at a k _assoc of about 1x 10.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that binds to human PD-L1 or PD-L2 at a k _dissoc of about 2 x 10 ^-5 1/s or slower, binds to human PD-1 at a k _dissoc of about 2.1 x 10 ^-5/s or slower, binds to human PD-1 at a k _dissoc of about 2.2 x 10 ^-5/s or slower, binds to human PD-1 at a k _dissoc of about 2.3 x 10 ^-5/s or slower, binds to human PD-1 at a k _dissoc of about 2.4 x 10 ^-5/s or slower, binds to human PD-1 at a k ^-5 of about 2.5 x 10 ^-5 1/s or slower, binds to human PD-1 at a k _dissoc of about 2.6 x 10 ^-5/s or slower, binds to human PD-1 at a k _dissoc of about 2.7 x 10 k 35/s or slower, binds to human PD-1 at a k _dissoc of about 2.4 x 10 k 35/s or slower, or a PD-3L 2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor, the PD-L1 and/or PD-L2 inhibitor blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 9nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 8nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 7nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 6nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4nM or less, blocks or inhibits the binding of human PD-L2 to human PD-1 with an IC50 of about 3nM or less than about 3nM or less, blocks or inhibits the binding of human PD-L2 to human PD-1 with an IC50 of about 6 or less than about 6nM or less than 2 is blocking or less than about 2 PD-1-2 binding of human PD-1 and human PD-1 with human PD-1 and human PD-1, or blocking human PD-1 or blocking human PD-L1 or human PD-L2 binding to human PD-1 with an IC50 of about 1nM or less.

In some embodiments, the PD-L1 inhibitor is dewaruzumab, also known as MEDI4736 (which is commercially available from Medimmune, LLC, a subsidiary of the netherlands, asbestrek) or an antigen-binding fragment, conjugate, or variant thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. patent No. 8,779,108 or U.S. patent application publication No. 2013/0034559, the disclosures of which are incorporated herein by reference. The clinical efficacy of Dewaruzumab has been described in Page et al, annual medical review, 2014,65,185-202; brahmer et al, journal of clinical oncology, 2014,32,5s (journal of journal 8021); and McDermott et al, comment on cancer treatment (CANCER TREATMENT Rev.), 2014,40,1056-64. The preparation and properties of Dewaruzumab are described in U.S. patent No. 8,779,108, the disclosure of which is incorporated herein by reference. The amino acid sequence of Dewaruzumab is set forth in Table 28. Dewaruzumab monoclonal antibodies include disulfide bonds at 22-96、22"-96"、23'-89'、23"'-89"'、135'-195'、135"'-195"'、148-204、148"-204"、215'-224、215"'-224"、230-230"、233-233"、265-325、265"-325"、371-429 and 371 "-429'; and N-glycosylation sites at Asn-301 and Asn-301'.

In some embodiments, the PD-L1 inhibitor comprises SEQ ID NO:178 and the heavy chain shown in SEQ ID NO: 179. In some embodiments, the PD-L1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO:178 and SEQ ID NO:179, or antigen binding fragments, fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 99% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 98% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 97% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 96% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 95% identity.

In some embodiments, the PD-L1 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of dewarfarin. In some embodiments, the PD-L1 inhibitor heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:180, the PD-L1 inhibitor light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:181 and conservative amino acid substitutions thereof. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:180 and SEQ ID NO:181 has at least 99% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:180 and SEQ ID NO:181 has at least 98% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:180 and SEQ ID NO:181 has at least 97% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:180 and SEQ ID NO:181 has at least 96% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:180 and SEQ ID NO:181 has at least 95% identity to the V _H region and the V _L region.

In some embodiments, the PD-L1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO: 182. SEQ ID NO:183 and SEQ ID NO:184 and the heavy chain CDR1, CDR2, and CDR3 domains of conservative amino acid substitutions thereof; and having the amino acid sequence of SEQ ID NO: 185. SEQ ID NO:186 and SEQ ID NO:187, and conservative amino acid substitutions thereof, light chain CDR1, CDR2, and CDR3 domains. In some embodiments, the antibody competes for binding to and/or to: the same epitope on PD-L1 as any of the antibodies previously described.

In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 biological analog monoclonal antibody approved by a drug administration reference to dewarfarin. In some embodiments, a biological analog comprises an anti-PD-L1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is dewarfarin. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is de-warfarin. anti-PD-L1 antibodies may be available to drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients included in a reference drug or reference biologic, wherein the reference drug or reference biologic is Devaluzumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients included in a reference drug or reference biologic, wherein the reference drug or reference biologic is Devaluzumab.

Table 28: reference to the amino acid sequence of a PD-L1 inhibitor of Dewaruzumab

In some embodiments, the PD-L1 inhibitor is avermectin, also known as MSB0010718C (commercially available from the merck group/smallpox) or an antigen binding fragment, conjugate, or variant thereof. The preparation and properties of avermectin are described in U.S. patent application publication No. US2014/0341917 A1, the disclosure of which is specifically incorporated herein by reference. The amino acid sequence of avermectin is set forth in table 29. Avalutamab has intra-heavy chain disulfide bonds (C23-C104) at 22-96, 147-203, 264-324, 370-428, 22"-96", 147"-203", 264"-324" and 370 "-428"; 22'-90', 138'-197', 22 '"-90'" and 138 '"-197'" of the sequence; 223-215 'and 223 "-215'" heavy-light chain intra-disulfide bonds (h 5-CL 126); heavy chain-heavy intra-chain disulfide bonds (h 11, h 14) at 229-229 "and 232-232"; 300. an N-glycosylation site at 300' (H CH 2N 84.4); fucosylation complex biantennary CHO-like glycans; and cleavage of the H CHS K2C-terminal lysine at 450 and 450'.

In some embodiments, the PD-L1 inhibitor comprises SEQ ID NO:188 and SEQ ID NO: 189. In some embodiments, the PD-L1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO:188 and SEQ ID NO:189, or an antigen binding fragment, fab fragment, single chain variable fragment (scFv), variant, or conjugate thereof. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:188 and SEQ ID NO:189 has at least 99% identity to the heavy and light chains. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:188 and SEQ ID NO:189 has at least 98% identity to the heavy and light chains. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:188 and SEQ ID NO:189 has at least 97% identity to the heavy and light chains. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:188 and SEQ ID NO:189 has at least 96% identity to the heavy and light chains. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:188 and SEQ ID NO:189 has at least 95% identity to the heavy and light chains.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or Variable Regions (VRs) of avermectin. In some embodiments, the PD-L1 inhibitor heavy chain variable region (VH) comprises SEQ ID NO:190, and the PD-L1 inhibitor light chain variable region (VL) comprises the sequence set forth in SEQ ID NO:191 and conservative amino acid substitutions thereof. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:190 and SEQ ID NO:191 has at least 99% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:190 and SEQ ID NO:191 has at least 98% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:190 and SEQ ID NO:191 has at least 97% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:190 and SEQ ID NO:191 has at least 96% identity to the V _H region and the V _L region. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:190 and SEQ ID NO:191 has at least 95% identity to the V _H region and the V _L region.

In some embodiments, the PD-L1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO: 192. SEQ ID NO:193 and SEQ ID NO:194 and the heavy chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 195. SEQ ID NO:196 and SEQ ID NO:197 and conservative amino acid substitutions thereof. In some embodiments, the antibody competes for binding to and/or to: the same epitope on PD-L1 as any of the antibodies previously described.

In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 biological analog monoclonal antibody that the drug administration references to avermectin approval. In some embodiments, a biological analog comprises an anti-PD-L1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is avermectin. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is avermectin. anti-PD-L1 antibodies may be available to drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is avermectin. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is avermectin.

Table 29: amino acid sequence of PD-L1 inhibitor of reference avermectin

In some embodiments, the PD-L1 inhibitor is atilizumab, also known as MPDL3280A or RG7446 (which is commercially available from the company barcello, switzerland, inc. Of the subsidiary gene texas TECENTRIQ), or an antigen-binding fragment, conjugate, or variant thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. patent No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. patent application publication nos. 2010/0203056A1, 2013/0045200A1, 2013/0045201A1, 2013/0045202A1, or 2014/0065135A1, the disclosures of which are specifically incorporated herein by reference. The preparation and properties of atilizumab are described in U.S. patent No. 8,217,149, the disclosure of which is incorporated herein by reference. The amino acid sequence of the atilizumab is set forth in table 30. The atilizumab has intra-heavy chain disulfide bonds (C23-C104) at 22-96, 145-201, 262-322, 368-426, 22"-96", 145"-201", 262"-322" and 368 "-426"; 23'-88', 134'-194', 23 '"-88'" and 134 '"-194'" of the light chain disulfide bond (C23-C104); 221-214 'and 221 "-214'" heavy-light chain intra-disulfide bonds (h 5-CL 126); heavy chain-heavy intra-chain disulfide bonds (h 11, h 14) at 227 "and 230-230"; and N-glycosylation sites at 298 and 298' (H CH2N84.4> A).

In some embodiments, the PD-L1 inhibitor comprises SEQ ID NO:198 and SEQ ID NO:199, a light chain as shown. In some embodiments, the PD-L1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO:198 and SEQ ID NO:199, or antigen binding fragments, fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:198 and SEQ id no:199 has heavy and light chains with at least 99% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:198 and SEQ ID NO:199 has a heavy and a light chain with at least 98% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:198 and SEQ ID NO:199 has a heavy and a light chain with at least 97% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:198 and SEQ ID NO:199 has a heavy and a light chain with at least 96% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:198 and SEQ ID NO:199 has heavy and light chains with at least 95% identity.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or Variable Regions (VRs) of atilizumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:200, and the PD-L1 inhibitor light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:201 and conservative amino acid substitutions thereof. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:200 and SEQ ID NO:201 has a V _H region and a V _L region of at least 99% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:200 and SEQ ID NO:201 has a V _H region and a V _L region of at least 98% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:200 and SEQ ID NO:201 has a V _H region and a V _L region of at least 97% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:200 and SEQ ID NO:201 has a V _H region and a V _L region of at least 96% identity. In some embodiments, the PD-L1 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:200 and SEQ ID NO:201 has a V _H region and a V _L region of at least 95% identity.

In some embodiments, the PD-L1 inhibitor comprises a polypeptide having the amino acid sequence of SEQ ID NO: 202. SEQ ID NO:203 and SEQ ID NO:204 and the heavy chain CDR1, CDR2, and CDR3 domains thereof with conservative amino acid substitutions; having the sequence of SEQ ID NO: 205. SEQ ID NO:206 and SEQ ID NO:207 and the light chain CDR1, CDR2 and CDR3 domains of conservative amino acid substitutions thereof. In some embodiments, the antibody competes for binding to and/or to: the same epitope on PD-L1 as any of the antibodies previously described.

In some embodiments, the anti-PD-L1 antibody is an anti-PD-L1 biological analog monoclonal antibody approved by a drug administration reference to atilizumab. In some embodiments, a biological analog comprises an anti-PD-L1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is an acti Li Zhushan antibody. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or application-authorized anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is an ati Li Zhushan antibody. anti-PD-L1 antibodies may be available to drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is an ati Li Zhushan antibody. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is an ati Li Zhushan antibody.

Table 30: reference to the amino acid sequence of a PD-L1 inhibitor of atilizumab

In some embodiments, PD-L1 inhibitors include those antibodies described in U.S. patent application publication No. US2014/0341917 A1, the disclosure of which is incorporated herein by reference. In other embodiments, antibodies that compete with any of these antibodies for binding to PD-L1 are also included. In some embodiments, the anti-PD-L1 antibody is MDX-1105, also known as BMS-935559, which is disclosed in U.S. patent No. 7,943,743, the disclosure of which is incorporated herein by reference. In some embodiments, the anti-PD-L1 antibody is selected from the anti-PD-L1 antibodies disclosed in U.S. patent No. 7,943,743, incorporated herein by reference.

In some embodiments, the PD-L1 inhibitor is a commercially available monoclonal antibody, e.g., INVIVOMAB anti-m-PD-L1 clone 10f.9g2 (catalog No. BE0101, biox Cell, inc., new hampson Xia Zhou parisons, usa). In some embodiments, the anti-PD-L1 antibody is a commercially available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH 1). A variety of commercially available anti-PD-L1 antibodies are known to those of ordinary skill in the art.

In some embodiments, the PD-L2 inhibitor is a commercially available monoclonal antibody, such as BIOLEGEND 24f.10c12 mouse IgG2a kappa isotype (catalog No. 329602, san diego bioleged, inc.), SIGMA anti-PD-L2 antibody (catalog No. SAB3500395, san lewis SIGMA oreq, missouri), or other commercially available anti-PD-L2 antibodies known to one of ordinary skill in the art.

2. Combination with CTLA-4 inhibitors

In some embodiments, the TIL therapy provided to the cancer patient can include treatment with a therapeutic TIL population alone, or can include combination treatment including TIL and one or more CTLA-4 inhibitors.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an immunoglobulin superfamily member and is expressed on the surface of helper T cells. CTLA-4 is a negative regulator of CD 28-dependent T cell activation and serves as a checkpoint for adaptive immune responses. Similar to the T cell costimulatory protein CD28, CTLA-4 binding antigen presents CD80 and CD86 on the cell. CTLA-4 delivers the inhibitor signal to T cells, while CD28 delivers the stimulation signal. Human antibodies to human CTLA-4 have been described as immunostimulating modulators of many disease states, for example, to treat or prevent viral and bacterial infections and to treat cancer (WO 01/14424 and WO 00/37504). A variety of fully human anti-human CTLA-4 monoclonal antibodies (mabs) have been studied in clinical trials for the treatment of various types of solid tumors, including, but not limited to, ipilimumab (MDX-010) and tremelimumab (CP-675,206).

In some embodiments, the CTLA-4 inhibitor may be any CTLA-4 inhibitor or CTLA-4 blocker known in the art. In particular, it is one of the CTLA-4 inhibitors or blockers described in more detail in the following paragraphs. With respect to CTLA-4 inhibitors, the terms "inhibitor," "antagonist," and "blocker" are used interchangeably herein. For the avoidance of doubt, references herein to CTLA-4 inhibitors as antibodies may refer to a compound or antigen-binding fragment, variant, conjugate or biological analogue thereof. For the avoidance of doubt, references herein to CTLA-4 inhibitors may also refer to small molecule compounds or pharmaceutically acceptable salts, esters, solvates, hydrates, co-crystals or prodrugs thereof.

CTLA-4 inhibitors suitable for use in the methods of the invention include, but are not limited to, anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (ipilimumab), tramadol, anti-CD 28 antibodies, anti-CTLA-4 adenetin, anti-CTLA-4 domain antibodies, single chain anti-a-4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, CTLA-4 inhibitors that agonize the co-stimulatory pathway, antibodies disclosed in PCT publication No. WO 2001/014424, antibodies disclosed in PCT publication No. WO 2004/035607, antibodies disclosed in us publication No. 2005/0201994, and antibodies disclosed in patent application EP 1212422B1, each of which are incorporated herein by reference. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227 and 6,984,720; PCT publication Nos. WO 01/14424 and WO 00/37504; and U.S. publication Nos. 2002/0039581 and 2002/086014, the disclosures of each of which are incorporated herein by reference. Other anti-CTLA-4 antibodies useful in the methods of the invention include, for example, antibodies disclosed in: WO 98/42752; U.S. Pat. nos. 6,682,736 and 6,207,156; hurwitz et al, proc. Natl. Acad.Sci.U.S. 95 (17): 10067-10071 (1998); camacho et al, journal of clinical oncology, 22 (145): abstract number 2505 (2004) (antibody CP-675206); mokyr et al, 58:5301-5304 (1998); and U.S. patent nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281, the disclosures of each of which are incorporated herein by reference.

Additional CTLA-4 inhibitors include (but are not limited to) the following: any inhibitor that is generally capable of disrupting the ability of CD28 antigen to bind to its cognate ligand, inhibiting CTLA-4 to bind to its cognate ligand, enhancing T cell responses through the costimulatory pathway, disrupting the ability of B7 to bind to CD28 and/or CTLA-4, disrupting the ability of B7 to activate the costimulatory pathway, disrupting the ability of CD80 to bind to CD28 and/or CTLA-4, disrupting the ability of CD80 to activate the costimulatory pathway, disrupting the ability of CD86 to bind to CD28 and/or CTLA-4, disrupting the ability of CD86 to activate the costimulatory pathway, and disrupting the costimulatory pathway, as a result of activation. This necessarily includes: small molecule inhibitors of CD28, CD80, CD86, CTLA-4, and other members of the costimulatory pathway; antibodies to CD28, CD80, CD86, CTLA-4, and other members of the costimulatory pathway; antisense molecules directed against CD28, CD80, CD86, CTLA-4, and other members of the costimulatory pathway; aldonin against CD28, CD80, CD86, CTLA-4 and other members of the costimulatory pathway; RNAi inhibitors (single and double stranded) of CD28, CD80, CD86, CTLA-4, and other members of the costimulatory pathway; and other CTLA-4 inhibitors.

In some embodiments, the CTLA-4 inhibitor binds CTLA-4 with a Kd of about 10 ^-6 M or less, 10 ^- ⁷ M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, 10 ^-12 M or less, e.g., between 10 ^-13 M and 10 ^-16 M, or within any range of any two of the foregoing values as endpoints. In some embodiments, the Kd of CTLA-4 inhibitor binding to CTLA-4 does not exceed 10-fold of the Kd of ipilimumab when compared using the same assay. In some embodiments, the Kd of CTLA-4 inhibitor binding to CTLA-4 is about the same as or less than (e.g., up to 10-fold lower or up to 100-fold lower than) the Kd of ipilimumab when compared using the same assay. In some embodiments, the CTLA-4 inhibitor inhibits binding of CTLA-4 to CD80 or CD86 by no more than 10-fold higher than the IC50 value of ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively, when compared using the same assay. In some embodiments, the CTLA-4 inhibitor inhibits binding of CTLA-4 to CD80 or CD86 by about the same or less (e.g., by up to 10-fold or up to 100-fold less) than the IC50 value of the ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively, when compared using the same assay.

In some embodiments, the CTLA-4 inhibitor is used in an amount sufficient to inhibit expression of CTLA-4 and/or reduce biological activity of CTLA-4 by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, e.g., between 50% and 75%, between 75% and 90%, or between 90% and 100%, relative to a suitable control. In some embodiments, the CTLA-4 pathway inhibitor is used in an amount sufficient to reduce the biological activity of CTLA-4 by reducing binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitable control, e.g., between 50% and 75%, between 75% and 90%, or between 90% and 100% relative to a suitable control. Suitable controls in the context of assessing or quantifying the effect of an agent of interest are typically comparable biological systems (e.g., cells or subjects) that have not been exposed to (or have been exposed to or treated with a negligible amount of) the agent of interest (e.g., CTLA-4 pathway inhibitor). In some embodiments, the biological system may serve as its own control, e.g., the biological system may be assessed prior to exposure to or treatment with an agent and compared to a state after the exposure or treatment is initiated or ended. In some embodiments, a history control may be used.

In some embodiments, the CTLA-4 inhibitor is ipilimumab (commercially available from bai meishi precious corporation as Yervoy) or a biological analog, antigen-binding fragment, conjugate, or variant thereof. As known in the art, ipilimumab refers to an anti-CTLA-4 antibody, a fully human IgG1 kappa antibody derived from a transgenic mouse having human genes encoding heavy and light chains to produce a functional human lineage. Ipilimumab can also be mentioned by its CAS accession number 477202-00-9 and in PCT publication No. WO 01/14424, which disclosures are incorporated by reference in their entirety for all purposes. Which is disclosed in the form of antibody 10 DI. In particular, ipilimumab contains a light chain variable region and a heavy chain variable region (having a light chain variable region comprising SEQ ID NO:211 and having a heavy chain variable region comprising SEQ ID NO: 210). Pharmaceutical compositions of ipilimumab include all pharmaceutically acceptable compositions comprising ipilimumab and one or more diluents, vehicles or excipients. Examples of pharmaceutical compositions containing ipilimumab are described in international patent application publication No. WO 2007/67959. Ipilimumab may be administered Intravenously (IV).

In some embodiments, the CTLA-4 inhibitor comprises SEQ ID NO:208 and SEQ ID NO: 209. In some embodiments, the CTLA-4 inhibitor comprises a peptide having the amino acid sequence of SEQ ID NO:208 and SEQ ID NO:209 or antigen binding fragments, fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:208 and SEQ ID NO:209 has at least 99% heavy and light chains. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:208 and SEQ ID NO:209 has at least 98% identity to the heavy and light chains. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:208 and SEQ ID NO:209 has at least 97% identity to the heavy and light chains. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:208 and SEQ ID NO:209 has at least 96% identity to the heavy and light chains. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:208 and SEQ ID NO:209 has at least 95% identity to the heavy and light chains.

In some embodiments, the CTLA-4 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of ipilimumab. In some embodiments, the CTLA-4 inhibitor heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:210, the CTLA-4 inhibitor light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:211 and conservative amino acid substitutions thereof. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:210 and SEQ ID NO:211 has at least 99% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:210 and SEQ ID NO:211 has at least 98% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:210 and SEQ ID NO:211 has at least 97% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:210 and SEQ ID NO:211 has at least 96% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:210 and SEQ ID NO:211 has at least 95% identity to the V _H region and the V _L region.

In some embodiments, the CTLA-4 inhibitor comprises a peptide having the amino acid sequence of SEQ ID NO: 212. SEQ ID NO:213 and SEQ ID NO:214 and the heavy chain CDR1, CDR2, and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 215. SEQ ID NO:216 and SEQ ID NO:217 and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains. In some embodiments, the antibody competes for binding to and/or to: CTLA-4 is identical to an epitope on any of the foregoing antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by a drug administration with reference to the volume of ipilimumab. In some embodiments, a biological analog comprises an anti-CTLA-4 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of ipilimumab is set forth in table 31. In some embodiments, the biological analog is an anti-CTLA-4 antibody that is authorized or filed authorized, wherein the anti-CTLA-4 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. anti-CTLA-4 antibodies can be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is ipilimumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is ipilimumab.

Table 31: amino acid sequence of ipilimumab

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In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, and the ipilimumab is administered at a dose of about 0.5mg/kg to about 10mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, and the ipilimumab is administered at the following doses: about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about 4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.5mg/kg, about 7mg/kg, about 7.5mg/kg, about 8mg/kg, about 8.5mg/kg, about 9mg/kg, about 9.5mg/kg or about 10mg/kg. In some embodiments, ipilimumab administration may also be initiated 1,2, 3,4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, wherein ipilimumab is administered at a dose of about 200mg to about 500mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, and the ipilimumab is administered at the following doses: about 200mg, about 220mg, about 240mg, about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg or about 500mg. In some embodiments, the ipilimumab administration is initiated 1,2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, and the ipilimumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, ipilimumab administration may also be initiated 1,2,3,4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, ipilimumab is administered to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab is administered at about mg/kg every 3 weeks for up to 4 doses to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab administration may also be initiated 1,2, 3, 4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, ipilimumab is administered to assist in the treatment of melanoma. In some embodiments, ipilimumab is administered at about 10mg/kg every 3 weeks for 4 doses, followed by 10mg/kg every 12 weeks for up to 3 years to aid in the treatment of melanoma. In some embodiments, ipilimumab administration may also be initiated 1,2, 3, 4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, ipilimumab is administered to treat advanced renal cell carcinoma. In some embodiments, ipilimumab is administered at about 1mg/kg every 3 weeks, followed by 3mg/kg of nivolumab on the same day for 4 doses to treat advanced renal cell carcinoma. In some embodiments, after completing the 4 doses of the combination, nivolumab may be administered as a single agent for advanced renal cell carcinoma and/or renal cell carcinoma according to standard dosing schedules. In some embodiments, ipilimumab administration may also be initiated 1,2,3,4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, ipilimumab is administered to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer. In some embodiments, ipilimumab is administered intravenously at about 1mg/kg every 3 weeks for 30 minutes followed by 3mg/kg nivolumab intravenously on the same day for 30 minutes for 4 doses to treat high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer. In some embodiments, after completing 4 doses of the composition, nivolumab is administered as a single agent for high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer as recommended according to standard dosing regimens. In some embodiments, ipilimumab administration may also be initiated 1,2,3, 4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, ipilimumab is administered to treat hepatocellular carcinoma. In some embodiments, ipilimumab is administered intravenously at about 3mg/kg every 3 weeks for 30 minutes followed by 1mg/kg nivolumab intravenously on the same day for 30 minutes for 4 doses to treat hepatocellular carcinoma. In some embodiments, after completing the 4 doses of the combination, nivolumab is administered as a single agent for hepatocellular carcinoma according to standard dosing protocols. In some embodiments, ipilimumab administration may also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1, 2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, ipilimumab is administered to treat metastatic non-small cell lung cancer. In some embodiments, ipilimumab is administered at about 1mg/kg every 6 weeks and nivolumab is administered at 3mg/kg every 2 weeks to treat metastatic non-small cell lung cancer. In some embodiments, ipilimumab is administered at about 1mg/kg every 6 weeks, plus 360mg nivolumab every 3 weeks with 2 cycles of platinum-containing dual chemotherapy to treat metastatic non-small cell lung cancer. In some embodiments, ipilimumab administration may also be initiated 1,2, 3, 4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, ipilimumab is administered to treat malignant pleural mesothelioma. In some embodiments, ipilimumab is administered at about 1mg/kg every 6 weeks and 360mg of nivolumab is administered every 3 weeks to treat malignant pleural mesothelioma. In some embodiments, ipilimumab administration may also be initiated 1,2, 3,4, or 5 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, ipilimumab administration may also be initiated 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

Trimemumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and CAS numbered 745013-59-6. Tramadol is disclosed in the form of antibody 11.2.1 in U.S. patent No. 6,682,736 (incorporated herein by reference). The amino acid sequences of the heavy and light chains of trimeumab are set forth in SEQ IND NO:218 and 219, respectively. Tramadol has been studied in clinical trials for the treatment of various tumors including melanoma and breast cancer; wherein the trimelimab is administered intravenously in single or multiple doses over 4 or 12 weeks in a dosage range between 0.01 and 15 mg/kg. In the regimen provided by the invention, the trimeumab is administered topically, in particular intradermally or subcutaneously. The effective amount of trimemab administered intradermally or subcutaneously is typically in the range of 5-200 mg/dose per person. In some embodiments, the effective amount of trimeumab is in the range of 10-150 milligrams per dose per person. In some particular embodiments, the effective amount of trimeumab is about 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175, or 200 milligrams per person.

In some embodiments, the CTLA-4 inhibitor comprises SEQ ID NO:218 and SEQ ID NO: 219. In some embodiments, the CTLA-4 inhibitor comprises a peptide having the amino acid sequence of SEQ ID NO:218 and SEQ ID NO:219 or antigen binding fragments, fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:218 and SEQ ID NO:219 has a heavy chain and a light chain having at least 99% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:218 and SEQ ID NO:219, the heavy and light chains having at least 98% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:218 and SEQ ID NO:219 has a heavy chain and a light chain having at least 97% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:218 and SEQ ID NO:219 has a heavy chain and a light chain having at least 96% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:218 and SEQ ID NO:219 has a heavy chain and a light chain having at least 95% identity.

In some embodiments, the CTLA-4 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of trimeumab. In some embodiments, the CTLA-4 inhibitor heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:220, the CTLA-4 inhibitor light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:221 and conservative amino acid substitutions thereof. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:220 and SEQ ID NO:221 has at least 99% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:220 and SEQ ID NO:221 has at least 98% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:220 and SEQ ID NO:221 has at least 97% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:220 and SEQ ID NO:221 has at least 96% identity to the V _H region and the V _L region. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:220 and SEQ ID NO:221 has at least 95% identity to the V _H region and the V _L region.

In some embodiments, the CTLA-4 inhibitor comprises a peptide having the amino acid sequence of SEQ ID NO: 222. SEQ ID NO:223 and SEQ ID NO:224 and the heavy chain CDR1, CDR2, and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 225. SEQ ID NO:226 and SEQ ID NO:227 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof. In some embodiments, the antibody competes for binding to and/or to: CTLA-4 is identical to an epitope on any of the foregoing antibodies.

In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 biological analog monoclonal antibody approved by a drug administration with reference to trimeumab. In some embodiments, a biological analog comprises an anti-CTLA-4 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is trimeumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequences of trimemab are shown in table 32. In some embodiments, the biological analog is an anti-CTLA-4 antibody that is authorized or filed authorized, wherein the anti-CTLA-4 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is trimeumab. anti-CTLA-4 antibodies can be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is trimeumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients contained in a reference drug or reference biologic, wherein the reference drug or reference biologic is trimeumab.

Table 32: amino acid sequence of trimeumab.

In some embodiments, the CTLA-4 inhibitor is trimelimab or a biological analog thereof, and the trimelimab is administered at a dose of about 0.5mg/kg to about 10mg/kg. In some embodiments, the CTLA-4 inhibitor is trimeumab or a biological analog thereof, and the trimeumab is administered at the following doses: about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about 4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.5mg/kg, about 7mg/kg, about 7.5mg/kg, about 8mg/kg, about 8.5mg/kg, about 9mg/kg, about 9.5mg/kg or about 10mg/kg. In some embodiments, administration of trimethoprim may also be initiated 1, 2,3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, administration of trimeumab can also begin 1, 2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the CTLA-4 inhibitor is trimelimab or a biological analog thereof, wherein the trimelimab is administered at a dose of about 200mg to about 500mg. In some embodiments, the CTLA-4 inhibitor is trimeumab or a biological analog thereof, and the trimeumab is administered at the following doses: about 200mg, about 220mg, about 240mg, about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg or about 500mg. In some embodiments, administration of trimeumab begins 1, 2, 3, 4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, administration of trimeumab can also begin 1, 2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the CTLA-4 inhibitor is trimeumab or a biological analog thereof, and the trimeumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, administration of trimethoprim may also be initiated 1,2, 3,4, or 5 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient). In some embodiments, administration of trimeumab can also begin 1,2, or 3 weeks prior to resection (i.e., prior to obtaining a tumor sample from a subject or patient).

In some embodiments, the CTLA-4 inhibitor is Zeff mab or a biological analog, antigen-binding fragment, conjugate, or variant thereof from Agenus. Zeff the mab is a fully human monoclonal antibody. Zeff Descriptors assigned Chemical Abstracts (CAS) accession numbers 2148321-69-9 and are also known as AGEN1884. Zeff preparation and characterization of mab is described in U.S. patent No. 10,144,779 and U.S. patent application publication No. US2020/0024350A1, the disclosures of which are incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor comprises SEQ ID NO:228 and SEQ ID NO: 229. In some embodiments, the CTLA-4 inhibitor comprises a peptide having the amino acid sequence of SEQ ID NO:228 and SEQ ID NO:229 or antigen binding fragments, fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 99% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 98% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 97% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 96% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:228 and SEQ ID NO:229 has heavy and light chains with at least 95% identity.

In some embodiments, the CTLA-4 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of Zeff mab. In some embodiments, the CTLA-4 inhibitor heavy chain variable region (V _H) comprises the amino acid sequence of SEQ ID NO:230, the CTLA-4 inhibitor light chain variable region (V _L) comprises the sequence set forth in SEQ ID NO:231 and conservative amino acid substitutions thereof. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:230 and SEQ ID NO:231 has a V _H region and a V _L region of at least 99% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:230 and SEQ ID NO:231 has a V _H region and a V _L region of at least 98% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:230 and SEQ ID NO:231 has a V _H region and a V _L region of at least 97% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:230 and SEQ ID NO:231 has a V _H region and a V _L region of at least 96% identity. In some embodiments, the CTLA-4 inhibitor comprises an amino acid sequence that is each identical to SEQ ID NO:230 and SEQ ID NO:231 has a V _H region and a V _L region of at least 95% identity.

In some embodiments, the CTLA-4 inhibitor comprises a peptide having the amino acid sequence of SEQ ID NO: 231. SEQ ID NO:233 and SEQ ID NO:234 and the heavy chain CDR1, CDR2, and CDR3 domains of conservative amino acid substitutions thereof; having the sequence of SEQ ID NO: 235. SEQ ID NO:236 and SEQ ID NO:237 and conservative amino acid substitutions thereof. In some embodiments, the antibody competes for binding to and/or to: CTLA-4 is identical to an epitope on any of the foregoing antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biological analog monoclonal antibody approved by the drug administration reference Zeff mab. In some embodiments, a biological analog comprises an anti-CTLA-4 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to an amino acid sequence of a reference drug or reference biological product, comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is Zeff lizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of Zeff mab is set forth in table 33. In some embodiments, the biological analog is an anti-CTLA-4 antibody that is authorized or filed authorized, wherein the anti-CTLA-4 antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, wherein the reference drug or reference biological product is Zeff lizumab. anti-CTLA-4 antibodies can be obtained from drug regulatory agencies such as the us FDA and/or eu EMA authorities. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients included in a reference drug or reference biologic, wherein the reference drug or reference biologic is Zeff lizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from excipients included in a reference drug or reference biologic, wherein the reference drug or reference biologic is Zeff lizumab.

Table 33: zeff amino acid sequence of Leizumab

Examples of additional anti-CTLA-4 antibodies include (but are not limited to): AGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659 and ADG116, which are known to those skilled in the art.

In some embodiments, the anti-CTLA-4 antibodies are anti-CTLA-4 antibodies ：US2019/0048096 A1;US2020/0223907;US2019/0201334;US2019/0201334;US 2005/0201994;EP 1212422 B1;WO 2018/204760;WO 2018/204760;WO 2001/014424;WO 2004/035607;WO 2003/086459;WO 2012/120125;WO 2000/037504;WO 2009/100140;WO 2006/09649;WO2005092380;WO 2007/123737;WO 2006/029219;WO 2010/0979597;WO 2006/12168; and WO1997020574 disclosed in any of the following patent publications, each of which is incorporated herein by reference. Additional CTLA-4 antibodies are described in the following: U.S. patent nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT publications WO 01/14424 and WO 00/37504; U.S. publication Nos. 2002/0039581 and 2002/086014; and/or U.S. patent nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281, each of which are incorporated herein by reference. In some embodiments, anti-CTLA-4 antibodies are those disclosed, for example, in the following: WO 98/42752; U.S. Pat. nos. 6,682,736 and 6,207,156; hurwitz et al, proc. Natl. Acad. Sci. USA, 1998,95,10067-10071 (1998); camacho et al, journal of clinical oncology 2004,22,145 (Abstract 2505 (2004) (antibody CP-675206), or Mokyr et al, cancer research (Cancer Res.)), 1998,58,5301-5304 (1998), each of which is incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996/040915 (incorporated herein by reference).

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, the anti-CTLA-4 RNAi molecules can be in the form of molecules described in: PCT publications WO 1999/032619 and WO 2001/029058; U.S. publication nos. 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, 2005/0100913, 2006/0024798, 2008/0050342, 2008/0081373, 2008/02485776, and 2008/055443; and/or U.S. Pat. nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecule is in the form of a double stranded RNAi molecule described in european patent No. EP 1309726 (incorporated herein by reference). In some cases, the anti-CTLA-4 RNAi molecules are in the form of double stranded RNAi molecules described in U.S. patent No. 7,056,704 and No. 7,078,196 (incorporated herein by reference). In some embodiments, the CTLA-4 inhibitor is an aptamer described in PCT publication No. WO 2004/081021 (incorporated herein by reference).

In other embodiments, the anti-CTLA-4 RNAi molecules of the invention are RNA molecules described in U.S. patent nos. 5,898,031, 6,107,094, 7,432,249 and 7,432,250, and european application No. EP 0928290 (incorporated herein by reference).

3. Pre-modulation of lymphocyte depletion in patients

In some embodiments, the invention includes a method of treating cancer with a population of TILs, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TILs according to the present disclosure. In some embodiments, the invention includes a population of TILs for treating cancer in a patient that has been pre-treated with non-myeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day 2 (days 27 and 26 before TIL infusion) and fludarabine 25mg/m ²/day 5 (days 27 to 23 before TIL infusion). In some embodiments, following non-myeloablative chemotherapy and TIL infusion according to the present disclosure (day 0), the patient receives intravenous infusion of IL-2 (aldinterleukin, commercially available as PROLEUKIN) intravenously at 720,000IU/kg every 8 hours to achieve physiologic tolerance. In certain embodiments, the TIL population is used in combination with IL-2 to treat cancer, wherein IL-2 is administered after the TIL population.

In general, lymphocyte depletion is achieved using administration of fludarabine or cyclophosphamide (active form called maphosamide) and combinations thereof. Such methods are described in Gassner et al, cancer immunology and immunotherapy 2011,60,75-85, muranski et al, natural clinical practice oncology 2006,3,668-681, dudley et al, journal of clinical oncology 2008,26,5233-5239 and Dudley et al, journal of clinical oncology 2005,23,2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL fludarabine. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL fludarabine. In some embodiments, fludarabine is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, fludarabine is administered at a dose of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4 to 5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4 to 5 days.

In some embodiments, the active form of cyclophosphamide is obtained by administering cyclophosphamide at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, the active form of cyclophosphamide is obtained by administering cyclophosphamide to a concentration of 1 μg/mL. In some embodiments, cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, cyclophosphamide is administered at a dose of 100mg/m ²/day, 150mg/m ²/day, 175mg/m ²/day, 200mg/m ²/day, 225mg/m ²/day, 250mg/m ²/day, 275mg/m ²/day, or 300mg/m ²/day. In some embodiments, the cyclophosphamide is administered intravenously (i.e., i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, cyclophosphamide treatment is administered intravenously at 250mg/m ²/day for 4 to 5 days. In some embodiments, cyclophosphamide treatment is administered intravenously at 250mg/m ²/day for 4 days.

In some embodiments, lymphocyte depletion is performed by administering fludarabine and cyclophosphamide together to the patient. In some embodiments, fludarabine is administered intravenously at 25mg/m ²/day and cyclophosphamide is administered intravenously at 250mg/m ²/day over 4 days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of 60mg/m ²/day for two days, followed by administration of fludarabine at a dose of 25mg/m ²/day for five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of 60mg/m ²/day for two days and fludarabine at a dose of 25mg/m ²/day for five days, wherein lymphocyte depletion is performed in a total of five days by administering both cyclophosphamide and fludarabine on the first two days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 50mg/m ²/day for two days and fludarabine at a dose of about 25mg/m ²/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and lymphocyte depletion is performed in a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 50mg/m ²/day for two days and fludarabine at a dose of about 20mg/m ²/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and lymphocyte depletion is performed in a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 40mg/m ²/day for two days and fludarabine at a dose of about 20mg/m ²/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and lymphocyte depletion is performed in a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 40mg/m ²/day for two days and fludarabine at a dose of about 15mg/m ²/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and lymphocyte depletion is performed in a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of 60mg/m ²/day and fludarabine at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for three days.

In some embodiments, cyclophosphamide is administered with mesna (mesna). In some embodiments, mesna is administered at 15 mg/kg. In some embodiments, mesna is infused, and if infused continuously, then mesna can be infused with cyclophosphamide over about 2 hours (day-5 and/or day-4) with the respective cyclophosphamide dose beginning, followed by infusion at a rate of 3 mg/kg/hour for the remaining 22 hours.

In some embodiments, lymphocyte depletion comprises the steps of: the patient is initially treated with the IL-2 regimen the next day after the third TIL population is administered to the patient.

In some embodiments, lymphocyte depletion comprises the steps of: patients were treated with an IL-2 regimen on the same day as the third TIL population was administered to the patients.

In some embodiments, lymphocyte depletion comprises 5 days of preconditioning therapy. In some embodiments, the number of days is indicated as from day-5 to day-1, or from day 0 to day 4. In some embodiments, the regimen comprises cyclophosphamide on days-5 and-4 (i.e., days 0 and 1). In some embodiments, the regimen comprises intravenous cyclophosphamide on days-5 and-4 (i.e., days 0 and 1). In some embodiments, the regimen comprises 60mg/kg intravenous cyclophosphamide on days-5 and-4 (i.e., days 0 and 1). In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the regimen further comprises fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine. In some embodiments, the regimen further comprises 25mg/m ² of intravenous fludarabine. In some embodiments, the regimen further comprises 25mg/m ² of intravenous fludarabine on day-5 and day-1 (i.e., day 0 to day 4). In some embodiments, the regimen further comprises 25mg/m ² of intravenous fludarabine on day-5 and day-1 (i.e., day 0 to day 4).

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day and fludarabine was administered at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for five days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day for two days, followed by fludarabine (fludarabine) at a dose of 25mg/m ²/day for five days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for three days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day and fludarabine was administered at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for three days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day and fludarabine was administered at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for one day.

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 34.

Table 34: exemplary lymphocyte depletion and treatment regimens

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In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 35.

Table 35: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60mg/kg

X

Mesna (as needed)

X

Fludarabine 25mg/m ²/day

X

TIL infusion

X

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 36.

Table 36: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60mg/kg

X

Mesna (as needed)

X

Fludarabine 25mg/m ²/day

X

TIL infusion

X

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 37.

Table 37: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 60mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 25mg/m ²/day			X	X	X
TIL infusion						X

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 38.

Table 38: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30mg/m ²/day	X	X	X	X	X
TIL infusion						X

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 39.

Table 38: exemplary lymphocyte depletion and treatment regimens

/>

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 40.

Table 40: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300mg/kg

X

Mesna (as needed)

X

Fludarabine 30mg/m ²/day

X

TIL infusion

X

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 41.

Table 41: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')	-5	-4	-3	-2	-1	0	1	2	3	4
											Cyclophosphamide 300mg/kg	X	X
Mesna (as needed)	X	X
											Fludarabine 30mg/m ²/day			X	X	X
TIL infusion						X

In some embodiments, the TIL infusion used with the foregoing embodiments of the myeloablative lymphocyte depletion regimen can be any of the TIL compositions described herein, as well as the addition of an IL-2 regimen and administration of a co-therapy (e.g., PD-1 and PD-L1 inhibitor) as described herein.

IL-2 protocol

In some embodiments, the IL-2 regimen comprises a high dose IL-2 regimen, wherein the high dose IL-2 regimen comprises an aldesleukin or a biological analog or variant thereof that is administered intravenously the next day after administration of the therapeutically effective portion of the therapeutic TIL population, the aldesleukin or biological analog or variant thereof being administered at a dose of 0.037mg/kg or 0.044mg/kg IU/kg (patient body weight) up to 14 doses per eight hours using 15 minute bolus intravenous infusion. After 9 days of rest, this time course can be repeated for a further 14 doses, up to 28 total doses. In some embodiments, IL-2 is administered in 1,2,3,4, 5, or 6 doses. In some embodiments, IL-2 is administered in a maximum dose of up to 6 doses.

In some embodiments, the IL-2 regimen comprises a decrementing IL-2 regimen. The decrementing IL-2 protocol has been described in O' Day et al, journal of clinical oncology 1999,17,2752-61 and Eton et al, cancer 2000,88,1703-9, the disclosures of which are incorporated herein by reference. In some embodiments, the decreasing IL-2 regimen comprises administering 18 x 10 ⁶IU/m² intravenously over 6 hours, then 18 x 10 ⁶IU/m² intravenously over 12 hours, then 18 x 10 ⁶IU/m² intravenously over 24 hours, then 4.5 x 10 ⁶IU/m² aldesleukin or a biological analog or variant thereof intravenously over 72 hours. This treatment cycle may be repeated every 28 days for up to four cycles. In some embodiments, the decrementing IL-2 regimen includes day 1 18,000,000IU/m ², day 29,000,000IU/m ², and day 3 and day 44,500,000IU/m ².

In some embodiments, the IL-2 regimen comprises a low dose IL-2 regimen. Any low dose IL-2 regimen known in the art may be used, including domiiguez-Villar and Hafler, nature immunology 2000,19,665-673; HARTEMANN et al, lancet Diabetes and Endocrinology (Lancet Diabetes Endocrinol.) 2013,1,295-305; and Rosenzwaig et al, the disclosure of which is incorporated herein by reference, the low dose IL-2 regimen described in annual rheumatism (Ann. Rheum. Dis.) 2019,78,209-217. In some embodiments, the low dose IL-2 regimen comprises 18 x 10 ⁶IU/m² of the aldesleukin or a biological analog or variant thereof, administered as a continuous infusion for 5 days every 24 hours; no IL-2 therapy was subsequently administered for 2 to 6 days; optionally followed by intravenous administration of the aldesleukin or a biological analog or variant thereof in the form of a continuous infusion of 18 x 10 ⁶IU/m² every 24 hours for a further 5 days; alternatively no IL-2 therapy is administered 3 weeks after that, followed by additional cycles of administration.

In some embodiments, IL-2 is administered in a maximum dose of up to 6 doses. In some embodiments, the high dose IL-2 regimen is suitable for pediatric use. In some embodiments, an aldesleukin of 600,000 International Units (IU)/kg is used every 8 to 12 hours up to 6 doses. In some embodiments, an aldesleukin of 500,000 International Units (IU)/kg is used every 8 to 12 hours up to 6 doses. In some embodiments, an aldesleukin of 400,000 International Units (IU)/kg is used every 8 to 12 hours up to 6 doses. In some embodiments, an aldesleukin of 500,000 International Units (IU)/kg is used every 8 to 12 hours up to 6 doses. In some embodiments, an aldesleukin of 300,000 International Units (IU)/kg is used every 8 to 12 hours dose for up to 6 doses. In some embodiments, an aldesleukin of 200,000 International Units (IU)/kg every 8 to 12 hours dose is used for up to 6 doses. In some embodiments, an aldesleukin of 100,000 International Units (IU)/kg is used every 8 to 12 hours dose for up to 6 doses.

In some embodiments, the IL-2 regimen comprises administering the pegylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering Bei Peia interleukin or a fragment, variant, or biological analog thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 regimen comprises administering THOR-707, or a fragment, variant, or biological analog thereof, at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4,6, 7, 14, or 21 days.

In some embodiments, the IL-2 regimen comprises administering nefiniujina or a fragment, variant or biological analog thereof after administration of the TIL. In certain embodiments, nefiniu is administered to the patient at a dose of 0.10 mg/day to 50 mg/day every 1,2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 regimen comprises administering an IL-2 fragment grafted onto the antibody backbone. In some embodiments, the IL-2 regimen comprises administering an antibody cytokine graft protein that binds to a low affinity receptor for IL-2. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (V _H) comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (V _L) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into the CDRs of V _H or V _L, wherein the antibody cytokine transplantation protein expands T effector cells in preference to regulatory T cells. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (V _H) comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (V _L) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into the CDRs of V _H or V _L, wherein the IL-2 molecule is a mutein and the antibody cytokine transplantation protein expands T effector cells in preference to regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody, or fragment, variant or biological analog thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 and SEQ ID NO:38 and a heavy chain selected from SEQ ID NO:37 and SEQ ID NO: 39.

In some embodiments, the serum half-life of the antibody cytokine transplantation proteins described herein is greater than that of a wild-type IL-2 molecule (e.g., without limitation, aldesleukinOr comparable molecules).

In some embodiments, the TIL infusion used with the foregoing embodiments of the myeloablative lymphocyte depletion regimen can be any of the TIL compositions described herein, which can also include MILs and PBL infusions in place of TIL infusions, as well as the addition of IL-2 regimens and administration of co-therapies (e.g., PD-1 and/or PD-L1 inhibitors and/or CTLA-4 inhibitors) as described herein.

5. Other methods of treatment

In other embodiments, the invention provides a method for treating a subject having cancer comprising administering to the subject a therapeutically effective dose of a therapeutic TIL population described in any of the preceding paragraphs as applicable above.

In other embodiments, the invention provides a method for treating a subject having cancer comprising administering to the subject a therapeutically effective dose of a TIL composition described in any preceding paragraph, as applicable above.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs that apply above, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administration of a therapeutically effective dose of a therapeutic TIL population as described in any of the preceding paragraphs that apply above or a therapeutically effective dose of a TIL composition as described in any of the preceding paragraphs that apply above.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs applicable above, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m 2/day for two days, followed by fludarabine at a dose of 25mg/m 2/day for five days.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, further comprising the step of beginning the treatment of the subject with a high dose IL-2 regimen the next day after administration of TIL cells to the subject.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs applicable above, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg as a 15 minute bolus intravenous infusion every eight hours until tolerized.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is a solid tumor.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any preceding paragraph as applicable above, wherein the cancer is melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), neuroglioblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs applicable above, wherein the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM) and gastrointestinal cancer.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is melanoma.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is HNSCC.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is cervical cancer.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is NSCLC.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is neuroglioblastoma (including GBM).

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is gastrointestinal cancer.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is a high mutation cancer.

In other embodiments, the invention provides a modified method for treating a subject having cancer as described in any of the preceding paragraphs as applicable above, wherein the cancer is pediatric hypermutated cancer.

In other embodiments, the invention provides a modified subject as applicable above for treating a subject having a cancer as described in any of the preceding paragraphs, wherein the cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple negative breast cancer), bone cancer, cancer caused by Human Papilloma Virus (HPV), central nervous system related cancers (including ependymoma, neural blastoma, neuroblastoma, pineal blastoma and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer and cervical adenocarcinoma), colorectal cancer, endometrial cancer, esophageal cancer, cancer at the esophageal-gastric junction, gastric cancer, gastrointestinal stromal tumor, neuroglioblastoma, glioma, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC), laryngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma and pharyngeal carcinoma), renal cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including uveal melanoma, choriocarcinoma or iris melanoma), mesothelioma (including malignant mesothelioma), ovarian cancer, pancreatic cancer (including ductal adenocarcinoma), penile carcinoma, rectal cancer, renal sarcoma, carcinoma, sarcoma, osteosarcoma, carcinoma of the thyroid gland (including sarcoma, carcinoma of the uterus, and other fibrosarcoma, and carcinoma).

In other embodiments, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs that apply above for use in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

In other embodiments, the invention provides a TIL composition as described in any preceding paragraph, as applicable above, for use in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In other embodiments, the invention provides a modified therapeutic TIL population as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administration of a therapeutically effective dose of the therapeutic TIL population as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above to the subject.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for five days.

In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above, further comprising the steps of: the patient is initially treated with the high dose IL-2 regimen the next day after TIL cells are administered to the patient.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs that apply above, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg in 15 minute bolus intravenous infusion every eight hours until tolerised.

In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above, wherein the cancer is a solid tumor.

In other embodiments, the invention provides a therapeutic TIL population as described in any preceding paragraph, or a modified TIL composition as described in any preceding paragraph, as applicable above, wherein the cancer is melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), neuroglioblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above, wherein the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In other embodiments, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above, wherein the cancer is melanoma.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is HNSCC.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is cervical cancer.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is NSCLC.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is neuroglioblastoma (including GBM).

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is gastrointestinal cancer.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is a hypermutated cancer.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is pediatric hypermutated cancer.

In other embodiments, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs that apply above or a modified TIL composition as described in any of the preceding paragraphs that apply above, wherein the cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple negative breast cancer), bone cancer, cancer caused by Human Papilloma Virus (HPV), central nervous system related cancers (including ependymoma, neural blastoma, neuroblastoma, pineal blastoma and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer and cervical adenocarcinoma), colorectal cancer, endometrial cancer, esophageal cancer, cancer at the esophageal-gastric junction, gastric cancer, gastrointestinal stromal tumor, neuroglioblastoma, glioma, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC), laryngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma and pharyngeal carcinoma), renal cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including uveal melanoma, choriocarcinoma or iris melanoma), mesothelioma (including malignant mesothelioma), ovarian cancer, pancreatic cancer (including ductal adenocarcinoma), penile carcinoma, rectal cancer, renal sarcoma, carcinoma, sarcoma, osteosarcoma, carcinoma of the thyroid gland (including sarcoma, carcinoma of the uterus, and other fibrosarcoma, and carcinoma).

In other embodiments, the invention provides the use of a therapeutic TIL population as described in any of the preceding paragraphs as applicable above in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

In other embodiments, the invention provides the use of a TIL composition as described in any of the preceding paragraphs as applicable above in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In other embodiments, the invention provides the use of a therapeutic TIL population as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above in a method of treating cancer in a subject, the method comprising administering to a subject a non-myeloablative lymphocyte depletion regimen, followed by administering to the subject a therapeutically effective dose of a therapeutic TIL population as described in any of the preceding paragraphs that apply above or a therapeutically effective dose of a TIL composition as described in any of the preceding paragraphs that apply above.

In other embodiments, the invention provides a modified therapeutic TIL population as described in any of the preceding paragraphs that apply above or a TIL composition as described in any of the preceding paragraphs that apply above, wherein a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administration of a therapeutically effective dose of the therapeutic TIL population or the therapeutically effective dose of the TIL composition to the subject.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m 2/day for two days, followed by fludarabine at a dose of 25mg/m 2/day for five days.

In other embodiments, the invention provides the use of a modified therapeutic TIL population as described in any of the preceding paragraphs as applicable above or the use of a TIL composition as described in any of the preceding paragraphs as applicable above, further comprising the steps of: starting on the day after administration of TIL cells to a patient, the patient is treated with a high dose IL-2 regimen.

In other embodiments, the invention provides for the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the high dose IL-2 regimen comprises administration of 600,000 or 720,000IU/kg in the form of 15 minute bolus intravenous infusions every eight hours until tolerated.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is a solid tumor.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition as described in any of the preceding paragraphs applicable above, wherein the cancer is melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), neuroglioblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is melanoma, HNSCC, cervical cancer, NSCLC, neuroglioblastoma (including GBM) and gastrointestinal cancer.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is melanoma.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is HNSCC.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is cervical cancer.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is NSCLC.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is neuroglioblastoma (including GBM).

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is gastrointestinal cancer.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is a high mutation cancer.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is pediatric hypermutated cancer.

In other embodiments, the invention provides the use of a modified therapeutic TIL population or TIL composition described in any of the preceding paragraphs as applicable above, wherein the cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple negative breast cancer), bone cancer, cancer caused by Human Papilloma Virus (HPV), central nervous system related cancers (including ependymoma, neural blastoma, neuroblastoma, pineal blastoma and primitive neuroectodermal tumors), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer and cervical adenocarcinoma), colorectal cancer, endometrial cancer, esophageal cancer, cancer at the esophageal-gastric junction, gastric cancer, gastrointestinal stromal tumor, neuroglioblastoma, glioma, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC), laryngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma and pharyngeal carcinoma), renal cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including uveal melanoma, choriocarcinoma or iris melanoma), mesothelioma (including malignant mesothelioma), ovarian cancer, pancreatic cancer (including ductal adenocarcinoma), penile carcinoma, rectal cancer, renal sarcoma, carcinoma, sarcoma, osteosarcoma, carcinoma of the thyroid gland (including sarcoma, carcinoma of the uterus, and other fibrosarcoma, and carcinoma).

Examples

Embodiments encompassed herein are now described with reference to the following examples. These examples are provided for illustrative purposes only and the present disclosure should in no way be construed as limited to these examples, but rather should be construed to cover any and all variations that become apparent based on the teachings provided herein.

Example 1: preparation of media for PRE-REP and REP procedures

This example describes a procedure for preparing tissue culture media suitable for protocols involving the culture of tumor-infiltrating lymphocytes (TILs) derived from various solid tumors. This medium can be used to prepare any of the TILs described in the present application and examples.

CM1 was prepared. The following reagents were removed from the freezer and allowed to warm in a 37 ℃ water bath: (RPMI 1640, human AB serum, 200mM L-glutamine). CM1 media was prepared by adding the ingredients to the top of a 0.2 μm filter unit appropriate for the volume to be filtered, according to table 42 below. Stored at 4 ℃.

Table 42: preparation of CM1

On the day of use, the required amount of CM1 was preheated in a 37℃water bath and 6000IU/mL of IL-2 was added.

Additional supplements may be provided as needed according to table 43.

Table 43: additional supplements to CM1 are required.

Preparation of CM2

The prepared CM1 is taken out from the refrigerator or fresh CM1 is prepared. Removal from refrigeratorAnd by mixing the prepared CM1 with an equal volume/>, in a sterile medium bottleTo prepare the desired amount of CM2. 3000IU/mL IL-2 was added to CM2 medium on the day of use. A sufficient amount of CM2 was made with 3000IU/mL IL-2 on the day of use. CM2 media flasks were labeled with name, manufacturer name abbreviation, filter/preparation date, expiration date for two weeks and stored at 4 ℃ before being needed for tissue culture.

Preparation of CM3

CM3 was prepared on the day of use. CM3The medium was the same, but 3000IU/mL IL-2 was supplemented on the day of use. CM3 was prepared in an amount that meets the experimental requirements by adding IL-2 stock directly to AIM-V bottles or bags. Thorough mixing was performed by gentle shaking. Immediately after addition of AIM-V, the bottles were labeled "3000IU/mL IL-2" and if excess CM3 was present, they were stored in the bottles at 4℃and labeled with the name of the medium, the abbreviation of the name of the manufacturer, the date on which the medium was prepared and its expiration date (7 days after preparation). After 7 days of storage at 4℃the medium supplemented with IL-2 was discarded.

Preparation of CM4

CM4 was identical to CM3, but was additionally supplemented with 2mM GlutaMAX ^TM (final concentration). 10mL of 200mM GlutamAX ^TM was added per 1L of CM3. CM4 was prepared in an amount meeting the experimental requirements by adding the IL-2 stock solution and the GlutaMAX ^TM stock solution directly to AIM-V bottles or bags. Thorough mixing was performed by gentle shaking. Immediately after addition to AIM-V, the bottles were labeled "3000IL/mL IL-2 and Glutamax". If excess CM4 is present, it is stored in a bottle at 4 ℃ and labeled with the medium name, "GlutaMAX" and its expiration date (7 days after preparation). After storage at 4℃for more than 7 days, the medium supplemented with IL-2 was discarded.

Example 2: use of IL-2, IL-15 and IL-21 cytokine mixtures

This example describes the use of IL-2, IL-15 and IL-21 cytokines as other T cell growth factors in combination with the TIL process of any of the examples herein.

Using the procedure described herein, TIL can grow from tumors in the presence of IL-2 in one group of experiments, with IL-2 replaced by a combination of IL-2, IL-15 and IL-21 in another group at the beginning of the culture. At the completion of prerep, cultures were assessed for expansion, phenotype, function (cd107 a+ and IFN- γ) and TCR vβ lineages. IL-15 and IL-21 are described elsewhere herein and in Santegoets et al, journal of transformation medicine (J.Transl. Med.), 2013,11,37.

The results may indicate that enhanced TIL expansion (> 20%) in CD4 ⁺ and CD8 ⁺ cells under IL-2, IL-15 and IL-21 treatment conditions can be observed relative to IL-2 alone. In TIL obtained from IL-2, IL-15 and IL-21 treated cultures, a major tilt of the CD8+ population occurred and the TCRVβ lineage was also tilted relative to IL-2 only cultures. IFN-. Gamma.and CD107a were elevated in IL-2, IL-15 and IL-21 treated TILs compared to IL-2 treated TILs alone.

Example 3: identification of individual batches of gamma irradiated peripheral monocytes

This example describes a simplified procedure for identifying a single batch of gamma irradiated peripheral mononuclear cells (PBMCs, also known as monocytes or MNCs) for use as allogeneic feeder cells in the exemplary methods described herein.

Each irradiated MNC feeder cell batch was prepared from a subject donor. In the presence of purified anti-CD 3 (clone OKT 3) antibodies and interleukin-2 (IL-2), separate screens were performed for the ability of each batch or donor to amplify TIL in REP. In addition, each batch of feeder cells was tested without the addition of TIL to verify that the gamma irradiation dose received was sufficient to render it replication incompetent.

The REP of TIL requires MNC feeder cells that are growth arrested by gamma irradiation. Membrane receptors on feeder cells MNC bind to anti-CD 3 (clone OKT 3) antibodies and crosslink with TIL in REP flasks, stimulating TIL expansion. Feeder cell batches were prepared by leukopenia from whole blood collected from subject donors. The leukopheresis product was centrifuged, washed, irradiated on Ficoll-Hypaque and cryopreserved under GMP conditions.

Importantly, patients receiving TIL therapy do not infuse live feeder cells, as this may lead to Graft Versus Host Disease (GVHD). Thus, growth of feeder cells is inhibited by gamma irradiation, resulting in double-stranded DNA breaks and loss of cell viability of MNC cells upon re-culture.

Feeder cell batches were evaluated according to two criteria: (1) Its ability to amplify TIL > 100-fold in co-culture, and (2) its replication incompetence.

Feeder cell batches were tested in mini-REP version using two major prerep TIL strains grown in vertical T25 tissue culture flasks. Feeder cell batches were tested against two different TIL strains, as each TIL strain was unique in its ability to proliferate in response to activation in REP. As a control, a number of irradiated MNC feeder cells, which have historically proven to meet the above criteria, were run with the test batches.

To ensure that all batches tested in a single experiment were subjected to equivalent testing, sufficient stock of the same prerep TIL strain could be used to test all conditions and all feeder cell batches.

For each batch of feeder cells tested, there were a total of six T25 flasks: pre-REP TIL strain #1 (2 flasks); pre-REP TIL strain #2 (2 flasks); and feeder cell control (2 flasks). Flasks containing TIL lines #1 and #2 were used to assess the ability of feeder cell batches to amplify TIL. Feeder control flasks were evaluated for replication incompetence of the feeder batch.

A. Experimental protocol

Day-2/3, the TIL strain was thawed. CM2 medium was prepared and CM2 was incubated in a 37℃water bath. 40mL of CM2 supplemented with 3000IU/mL IL-2 was prepared. Keep warm until use. 20mL of preheated CM2 without IL-2 was placed in each of two 50mL conical tubes, labeled with the name of the TIL strain used. Two designated prerep TIL strains were removed from LN2 storage and vials transferred to the tissue culture chamber. The vials were thawed by placing them in a sealed zipper storage bag in a 37 ℃ water bath until a small amount of ice remained.

The contents of each vial were immediately transferred to 20mL CM2 in the prepared labeled 50mL conical tube using a sterile pipette. The cells were washed with CM2 supplemented to 40mL without IL-2 and centrifuged at 400 XCF for 5 min. The supernatant was aspirated and resuspended in 5mL warmed CM2 supplemented with 3000IU/mL IL-2.

Small aliquots (20 μl) were removed in duplicate for cell counting using an automated cell counter. The count is recorded. At the time of counting, 50mL conical tubes with TIL cells were placed in a 37 ℃,5% CO ₂ wet incubator, and the lid was released to allow gas exchange. Cell concentration was measured and TIL was diluted to 1X 10 ⁶ cells/mL in CM2 supplemented with 3000IU/mL IL-2.

Cultures were performed at 2 ml/well in multiple wells of a 24-well tissue culture plate, as needed, in a 37 ℃ wet incubator until day 0 of mini REP. Different TIL strains were grown in separate 24-well tissue culture plates to avoid confusion and potential cross-contamination.

Day 0, mini REP was started. Sufficient CM2 medium was prepared for the number of feeder cell batches to be tested. (for example, to test 4 feeder cell batches at a time, 800mL CM2 medium was prepared). A portion of the CM2 prepared above was aliquoted, supplemented with 3000IU/mL IL-2 for cell culture. (e.g., to test 4 feeder cell batches at a time, 500mL CM2 medium with 3000IU/mL IL-2 was prepared).

The 24-well plates with TIL cultures were removed from the incubator and transferred to BSC with each TIL line independently operated to prevent cross contamination.

About 1mL of medium was removed from each well of the TIL to be used using a sterile pipette or 100-1000 μl pipette and tip and placed into the unused wells of a 24-well tissue culture plate.

The remaining medium was mixed with TIL in the wells using a fresh sterile pipette or 100-1000 μl pipette and tip to re-suspend the cells, and then the cell suspension was transferred to a 50mL conical tube labeled with the TIL lot name and the volume was recorded.

The wells were washed with the remaining medium and the volumes transferred to the same 50mL conical tube. The cells were spun at 400 XCF to collect cell pellet. The culture supernatant was aspirated and the cell pellet was resuspended in 2 to 5mL CM2 medium containing 3000IU/mL IL-2, the volume used being based on the number of wells collected and the size of the pellet, i.e. the volume should be sufficient to ensure a concentration > 1.3x10 ⁶ cells/mL.

The cell suspension was thoroughly mixed using a serum pipette and the volume was recorded. 200 μl was removed for cell counting using an automated cell counter. At the time of counting, 50mL conical tubes containing TIL cells were placed in a 5% CO ₂, 37 ℃ wet incubator, and the lid was released to allow gas exchange. The count is recorded.

A50 mL conical tube containing TIL cells was removed from the incubator and the cells were resuspended in warmed CM2 supplemented with 3000IU/mL IL-2 at a concentration of 1.3X10 ⁶ cells/mL. The 50mL conical tube was returned to the incubator and the lid was released.

The above steps are repeated for the second TIL strain.

Just prior to inoculation of the TIL into the T25 flask used for the experiment, the TIL was diluted 1:10 to a final concentration of 1.3 x 10 ⁵ cells/mL as shown below.

MACS GMP CD3 a 3 pure (OKT 3) working solution was prepared. Stock solution of OKT3 (1 mg/mL) was taken out from the refrigerator at 4℃and placed in BSC. OKT3 was used in medium with a final concentration of 30ng/mL in micro REP.

600Ng OKT3 was required per 20mL in each T25 flask used for the experiment; this corresponds to 60. Mu.L of 10. Mu.g/mL solution per 20mL or 360. Mu.L for all 6 test flasks per feeder cell batch.

For each feeder cell batch tested, 400. Mu.L of 1mg/mL OKT3 at a 1:100 dilution was prepared at a working concentration of 10. Mu.g/mL (e.g., 4 feeder cell batches were tested at a time, 1600. Mu.L of 1mg/mL OKT3 at a 1:100 dilution was prepared: 16. Mu.L of 1mg/mL OKT3+1.584mL of CM2 medium with 3000IU/mL IL-2).

T25 flasks were prepared. Each flask and filled flask was labeled with CM2 medium prior to the preparation of feeder cells. The flask was placed in a 5% CO ₂ wet incubator at 37℃to keep the medium warm while waiting for the rest of the components to be added. After the preparation of feeder cells, the components were added to CM2 in each flask.

Other information is provided in table 44.

TABLE 44 solution information

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Feeder cells were prepared. For this protocol, a minimum of 78×10 ⁶ feeder cells were required per batch tested. Each 1mL vial frozen by SDBB had 100X 10 ⁶ viable cells when frozen. Assuming a recovery of 50% after thawing from liquid N ₂ storage, it is recommended to thaw at least two 1mL vials of feeder cells per batch, thus using an estimated 100X 10 ⁶ viable cells in each REP. Or if supplied in 1.8mL vials, only one vial provides enough feeder cells.

Before thawing the feeder cells, about 50mL of CM2 free of IL-2 was pre-warmed for each feeder cell batch to be tested. The designated feeder cell batch vials were removed from the LN2 reservoir, placed in a zipper storage bag, and placed on ice. The vials were thawed in closed zipper storage bags by immersion in a 37 ℃ water bath. The vials were removed from the zipper pack, sprayed or wiped with 70% etoh, and transferred to the BSC.

The contents of the feeder cell vials were immediately transferred to 30mL of warmed CM2 in a 50mL conical tube using a pipette. The vials were washed with a small volume of CM2 to remove any residual cells in the vials and centrifuged at 400 xcf for 5 minutes. The supernatant was aspirated and resuspended in 4mL warmed CM2 plus 3000IU/mL IL-2. 200 μl was removed for cell counting using an automated cell counter. The count is recorded.

Cells were resuspended at 1.3X10 ⁷ cells/mL in warm CM2 plus 3000IU/mL IL-2. TIL cells were diluted from 1.3×10 ⁶ cells/mL to 1.3×10 ⁵ cells/mL.

Co-cultures were established. TIL cells were diluted from 1.3×10 ⁶ cells/mL to 1.3×10 ⁵ cells/mL. 4.5mL of CM2 medium was added to a 15mL conical tube. TIL cells were removed from the incubator and resuspended well using a 10mL serum pipette. 0.5mL of cells were removed from the 1.3X10 ⁶ cells/mLTIL suspension and added to 4.5mL of medium in a 15mL conical tube. The TIL stock solution bottles were returned to the incubator. Thoroughly mixed. The second TIL strain was repeated.

Flasks containing pre-warmed medium were transferred from the incubator to the BSC for single feeder cell batches. Feeder cells were mixed by pipetting up and down several times with a 1mL pipette tip, and then 1mL (1.3x10 ⁷ cells) was transferred to each flask for the feeder cell batch. To each flask was added 60. Mu.L of OKT3 working stock solution (10. Mu.g/mL). Two control bottles were returned to the incubator.

1ML (1.3X10 ⁵) of each TIL batch was transferred to a correspondingly labelled T25 flask. The flask was returned to the incubator and vertically cultured. Intervention was started until day 5. This procedure was repeated for all feeder cell batches tested.

On day 5, the medium was changed. CM2 was prepared with 3000IU/mL IL-2. Each flask required 10mL. 10mL warmed CM2 with 3000IU/mL IL-2 was transferred to each flask by a 10mL pipette. The flask was returned to the incubator and cultivated upright until day 7. All feeder cell batches tested were repeated.

Day 7, collection. The flask was removed from the incubator and transferred to the BSC taking care not to disturb the cell layer at the bottom of the flask. 10mL of medium was removed from each test flask and 15mL of medium was removed from each control flask without disturbing the cells growing on the bottom of the flask.

Cells were resuspended in the remaining medium using a 10mL serum pipette and thoroughly mixed to break up any clumps of cells. After thoroughly mixing the cell suspension by pipetting, 200 μl was removed for cell counting. TIL is counted using appropriate standard procedures in conjunction with an automatic cytometer device. The count was recorded on day 7. This procedure was repeated for all feeder cell batches tested.

Replication incompetence of feeder control flasks was assessed, and fold expansion of TIL-containing flasks was assessed starting on day 0.

On day 7, feeder cell control flasks continued to day 14. After completion of the counting of feeder control flasks on day 7, 15mL of fresh CM2 medium containing 3000IU/mL IL-2 was added to each of the control flasks. The control flask was returned to the incubator and incubated in an upright position until day 14.

On day 14, the non-proliferating feeder control flask was extended. The flask was removed from the incubator and transferred to the BSC taking care not to disturb the cell layer at the bottom of the flask. Approximately 17mL of medium was removed from each control bottle without disturbing the cells growing at the bottom of the bottle. Cells were resuspended in medium remaining in the medium using a 5mL serum pipette and thoroughly mixed to break up any clumps of cells. The volume of each flask was recorded.

After thoroughly mixing the cell suspension by pipetting, 200 μl was removed for cell counting. TILs were counted and counts recorded using appropriate standard operating procedures in conjunction with an automatic cytometer device. This procedure was repeated for all feeder cell batches tested.

B. results and acceptance criteria schemes

As a result. The dose of gamma irradiation is sufficient to render the feeder cells replication incompetent. All batches were expected to meet the evaluation criteria and also showed a decrease in total viable cell number of feeder cells remaining on day 7 of REP culture compared to day 0. It is expected that on day 7 of REP culture, all feeder cell batches will meet the 100-fold increase in TIL rating criteria. It is expected that day 14 feeding control bottle counts will continue with the non-proliferative trend seen on day 7.

Acceptance criteria. Duplicate TIL strains tested for each batch of feeder cells met the following acceptance criteria. The acceptance criteria are doubled as shown in table 45 below.

Table 45. Acceptance criteria embodiments of criteria.

When incubated in the presence of 30ng/mL OKT3 antibody and 3000IU/mL IL-2, it was assessed whether the dose of irradiation was sufficient to render MNC feeder cells replication incompetent. Replication incompetence was assessed as by total viable cell count (TVC) measured by automated cell count on day 7 and day 14 of REP.

Acceptance criteria were "no growth" meaning that the initial number of living cells put into culture from day 0 of REP did not increase in total number of living cells on days 7 and 14.

Feeder cells were assessed for their ability to support TIL expansion. TIL growth was measured as fold expansion of living cells from day 0 of REP to day 7 of REP. At day 7, the TIL cultures achieved a minimum of 100-fold expansion (i.e., over 100-fold the total number of viable TIL cells put into culture at day 0 of REP) as assessed by automated cell counting.

Emergency testing of MNC feeder cell batches that do not meet acceptance criteria. In case the MNC feeder cell batch does not meet any of the acceptance criteria outlined above, the following steps will be performed to retest the batch to exclude simple experimenter errors as its cause.

If there are more than two remaining satellite test vials (SATELLITE TESTING VIAL) for the batch, the batch is retested. If a lot has one or no remaining satellite test vials, the lot is not acceptable according to the acceptance criteria listed above.

In order to be acceptable, the batch in question and the control batch must meet the above acceptance criteria. After meeting these criteria, the batch is permitted to be used.

Example 4: preparation of IL-2 stock solutions

This example describes the process of solubilizing purified lyophilized recombinant human interleukin-2 in stock samples suitable for use in other tissue culture protocols, including all those described in the present application and examples, including those involving the use of rhIL-2.

And (5) program. A0.2% acetic acid solution (HAc) was prepared. 29mL of sterile water was transferred to a 50mL conical tube. To a 50mL conical tube was added 1mL 1N acetic acid. Thorough mixing was performed by inverting the tube 2 to 3 times. The HAc solution was sterilized by filtration using a Steriflip filter.

PBS containing 1% HAS was prepared. In a 150mL sterile filter unit, 4mL of 25% HSA stock solution was added to 96mL of PBS. The solution was filtered. Stored at 4 ℃. The form was filled out for each vial of rhIL-2 prepared.

A rhIL-2 stock solution (6X 10 ⁶ IU/mL final concentration) was prepared. The rhIL-2 is different for each batch, and the information required is found in the manufacturer's analytical Certificate (COA), for example: 1) mass per vial of rhIL-2 (mg), 2) specific activity of rhIL-2 (IU/mg) and 3) recommended 0.2% HAc recovery volume (mL).

The volume of 1% HSA required for the rhIL-2 batch was calculated using the following formula:

For example, according to COA of rhIL-2 batch 10200121 (Cellgenix), the specific activity of a 1mg vial was 25X 10 ⁶ IU/mg. It is recommended to restore rhIL-2 in 2mL of 0.2% HAc.

The rubber stopper of the IL-2 vial was wiped with an alcohol wipe. Using a 16G needle connected to a 3mL syringe, 0.2% of the recommended volume of HAc was injected into the vial. Care should be taken not to remove the stopper when the needle is withdrawn. The vial was inverted 3 times and swirled until all the powder was dissolved. Carefully remove the stopper and rest on the alcohol wipe. Adding the calculated volume to the vial

1％HSA。

The rhIL-2 solution was stored. For short term storage (< 72 hours), vials were stored at 4 ℃. For long term storage (> 72 hours), the vials were aliquoted into smaller volumes and stored in frozen vials at-20 ℃ prior to being ready for use. Avoiding freeze/thaw cycles. Expiration was 6 months after the date of preparation. Rh-IL-2 tags included vendor and catalog numbers, lot numbers, expiration dates, operator acronyms, concentrations, and aliquot volumes.

Example 5: cryopreservation process

This example describes a cryopreservation process method for TIL prepared according to the procedures described herein using a model 7454 CryoMed controlled rate freezer (Thermo Scientific).

The equipment used was as follows: aluminum cartridge holders (compatible with CS750 freezer bags), freezer boxes for 750mL bags, low pressure (22 psi) liquid nitrogen tanks, refrigerators, thermocouple sensors (ribbon bags), and CryoStore CS750 freezer bags (OriGen Scientific).

The freezing process provides a rate of 0.5 ℃ from nucleation to-20 ℃ and a cooling rate of 1 ℃/min to-80 ℃ end temperature. The program parameters were as follows: step 1: waiting at 4deg.C; step 2:1.0 ℃/min (sample temperature) to-4 ℃; step 3:20.0 ℃/min (box temperature) reaches-45 ℃; step 4:10.0 ℃/min (box temperature) to-10.0 ℃; step 5:0.5 ℃/min (box temperature) reaches-20 ℃; and step 6: 1.0deg.C/min (sample temperature) to-80deg.C.

Example 6: tumor amplification procedure Using defined Medium

The above disclosed process can be performed with the corresponding defined media (e.g., CTS ^TMOpTmizer^TM T cell expanded SFM, sammer femto, including, e.g., DM1 and DM 2) instead of CM1 and CM2 media.

Example 7: exemplary GEN 2 preparation for cryopreserved TIL cell therapies

This example describes cGMP manufacture of a TIL cell therapy procedure of the eikovans biotherapy company in G-Rex flasks according to current organization good operating specifications (current Good Tissue Practices) and current good manufacturing specifications (current Good Manufacturing Practices). This example describes an exemplary cGMP manufacture for performing a TIL cell therapy procedure in G-Rex flasks according to current organization good specifications and current good manufacturing specifications.

Table 46: the process augments the exemplary plan.

Table 47: culture flask volume

On day 0, CM1 medium was prepared. In BSC, reagents were added to RPMI 1640 medium bottles. The following reagents t were added per bottle: heat-inactivating human AB serum (100.0 mL); glutaMax ^TM (10.0 mL); gentamicin sulfate, 50mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL).

Unnecessary material is fetched from the BSC. Media reagents were dispensed from BSC, and gentamicin sulfate and HBSS were retained at BSC for formulation of wash media preparation.

An aliquot of IL-2 was thawed. A1.1 mL aliquot of IL-2 (6X 10 ⁶ IU/mL) (BR 71424) was thawed until all ice had melted. Record IL-2: lot number and expiration date.

IL-2 stock was transferred to medium. In BSC, 1.0mL of IL-2 stock solution was transferred to a prepared CM1 day 0 medium bottle. 1 bottle of CM1 day 0 medium and 1.0mL of IL-2 (6X 10 ⁶ IU/mL) were added.

The G-REX100MCS is delivered to the BSC. The G-REX100MCS (W3013130) is aseptically delivered to the BSC.

All complete CM1 day 0 media was pumped into G-REX100MCS flasks. Tissue fragment conical tube or G-REX100MCS.

Tumor wash medium preparation on day 0. In BSC, 5.0mL gentamicin (W3009832 or W3012735) was added to a 1X 500mL HBSS media (W3013128) bottle. Each bottle is added with: HBSS (500.0 mL); gentamicin sulfate, 50mg/mL (5.0 mL). Filtered HBSS containing gentamicin was prepared by a 1l 0.22 micron filter unit (W1218810).

Tumor treatment on day 0. Tumor samples were obtained and immediately transferred to a kit at 2-8 ℃ for processing. Tumor wash medium was aliquoted. Tumor wash 1 was performed using 8 "forceps (W3009771). Tumors were removed from the flask and transferred to prepared "wash 1" dishes. This is followed by tumor wash 2 and tumor wash 3. Tumors were measured and evaluated. It was assessed whether >30% necrosis and/or adipose tissue was observed throughout the tumor area. The anatomy is cleaned when applicable. If the tumor is large and >30% tissue appearance necrosis/fat is observed, "clean-up segmentation" is performed by removing necrotic/adipose tissue using a combination of scalpels and/or forceps while preserving tumor internal structure. Tumors were dissected. Using a combination of scalpels and/or forceps, the tumor specimen is cut into an even number of appropriately sized pieces (up to 6 intermediate pieces). Intermediate tumor fragments were metastasized. Dividing tumor fragments into sizes approximately 3 x 3mm blocks. The intermediate chips are stored to prevent dehydration. The intermediate fragment segmentation is repeated. The number of collected blocks is measured. If only the desired tissue is retained, additional beneficial tumor fragments are selected from the "beneficial intermediate fragment" 6-well plate to fill up to 50 pieces of droplets.

A conical tube was prepared. Tumor fragments were transferred to a 50mL conical tube. Prepare the BSC for G-REX100MCS. The G-REX100MCS was removed from the incubator. G-REX100MCS flasks were aseptically transferred to BSC. Tumor fragments were added to G-REX100MCS flasks. So that the fragments are uniformly distributed.

The G-REX100MCS was incubated according to the following parameters: the G-REX flask was incubated: temperature LED display: 37.0+/-2.0 ℃; CO ₂ percent: 5.0+ -1.5% CO ₂. And (3) calculating: the heat preservation time; lower limit = incubation time +252 hours; upper limit = incubation time +276 hours.

After the process was completed, all remaining warmed medium was discarded and an aliquot of IL-2 was thawed.

Day 11-preparation of culture medium monitoring incubator. Incubator parameters: temperature LED display: 37.0+/-2.0 ℃; percentage of CO 2: 5.0+ -1.5% CO2.

A3X 1000mL RPMI 1640 medium (W3013112) flask and a 3X 1000mL AIM-V (W3009501) flask were warmed for 30 minutes or more in an incubator. The RPMI 1640 medium flasks were removed from the incubator. RPMI 1640 medium flasks were prepared. The medium was filtered. An aliquot of 3X 1.1mL IL-2 (6X 106 IU/mL) was thawed (BR 71424). AIM-V medium was removed from the incubator. IL-2 was added to AIM-V. The 10L Labtainer bag and relay pump transfer unit were aseptically transferred to the BSC.

10L Labtainer bags of medium were prepared. The Baxa pump was prepared. 10L Labtainer bags of medium were prepared. The medium was pumped into 10L Labtainer. The automatic pump pipette is removed from the Labtainer bag.

The medium was mixed. Gently knead the bag for mixing. The medium is sampled according to a sampling plan. 20.0mL of the medium was removed and placed in a 50mL conical tube. Cell count dilution tubes were prepared. In BSC, 4.5mL AIM-V medium labeled "for cell count dilution" and lot number was added to four 15mL conical tubes. The reagent was transferred from the BSC to 2 to 8 ℃. A 1L transfer packet is prepared. Outside the BSC, 1L transfer packets were welded (5.11 according to process notes) to a transfer device attached to a prepared "complete CM2 day 11 medium" bag. Feeder cells transfer bags were prepared. Complete CM2 day 11 medium was incubated.

Day 11-TIL pretreatment tables were collected. Incubator parameters: temperature LED display: 37.0+/-2.0 ℃; CO ₂ percent: 5.0+ -1.5% CO ₂. The G-REX100MCS was removed from the incubator. Prepare 300mL transfer pack. The transfer packets were welded to G-REX100MCS.

The flask for TIL collection was prepared and TIL collection was started. TIL was collected. Using GatheRex, the cell suspension was transferred through a hemofilter into a 300mL transfer bag. Adherent cells on the membrane were examined.

The flask membrane was rinsed. The clip on the G-REX100MCS is closed. Ensure that all clips are closed. Heat seal TIL and "supernatant" transfer packets. The volume of the TIL suspension was calculated. The supernatant transfer pack was prepared for sampling.

Bac-T samples were taken. In the BSC, about 20.0mL of supernatant was aspirated from a 1L "supernatant" transfer bag and dispensed into a sterile 50mL conical tube.

Inoculation BacT is performed according to the sampling plan. 1.0mL of the sample was removed from the prepared 50mL conical tube labeled BacT using a syringe of appropriate size and inoculated into an anaerobic jar.

Incubating the TIL. The TIL transfer packs were placed in an incubator before needed. Cell counting and calculation were performed. The average value of the living cell concentration and the average value of the survival rate of the cells subjected to cell counting were measured. Survival rate ≡2. Living cell concentration ≡2. The upper and lower limits of the count are determined. Lower limit: mean viable cell concentration x 0.9. Upper limit: viable cell concentration was mean x 1.1. Confirm that both counts are within acceptable limits. The average viable cell concentration was determined from all four counts performed.

Adjusting the volume of the TIL suspension: the adjusted volume of TIL suspension after removal of the cell count sample was calculated. Total TIL cell volume (a). Volume of cell count samples taken (4.0 mL) (B) adjusted TIL cell total volume c=a-B.

The total number of viable TIL cells was calculated. Average viable cell concentration: total volume; total number of living cells: c=a×b.

Calculation of flow cytometry: if the total count of viable TIL cells is ≡4.0X10 ⁷, the volume of 1.0X10 ⁷ cells obtained for the flow cytometry samples is calculated.

Total number of living cells required for flow cytometry: 1.0X10 ⁷ cells. Cell volume required for flow cytometry: viable cell concentration divided by 1.0X10 ⁷ cells A.

The volume of the TIL suspension was calculated to be equal to 2.0×10 ⁸ living cells. The excess TIL cell volume to be removed is calculated as needed, excess TIL is removed and the TIL is placed in an incubator as needed. The total amount of excess TIL removed as needed was calculated.

The calculated amount of CS-10 medium added to the excess TIL cells at the target cell concentration for freezing was 1.0X10 ⁸ cells/mL. Excess TIL was centrifuged as needed. The conical tube was observed and CS-10 was added.

Fill the vial. 1.0mL of the cell suspension was aliquoted into appropriately sized frozen vials. The remaining volume was aliquoted into appropriately sized frozen vials. If the volume is less than or equal to 0.5mL, CS10 is added to the vial until the volume is 0.5mL.

The cell volume required to obtain 1 x 10 ⁷ cells for cryopreservation was calculated. Samples were removed for cryopreservation. The TIL was placed in an incubator.

And (5) freezing and preserving the sample. The conical tube was observed and the CS-10 was slowly added and the volume of 0.5mL of CS10 added was recorded.

Day 11-feeder cells. Feeder cells were obtained. At least two different batches of 3 bags of feeder cells were obtained from an LN2 freezer. Cells were stored on dry ice prior to preparation for thawing. A water bath or cryotherm is prepared. The feeder cells were thawed at 37.0.+ -. 2.0 ℃ water bath or cytotherm min or until just the ice had disappeared. The medium was removed from the incubator. The thawed feeder cells were pooled. Feeder cells were added to the transfer bag. Feeder cells were dispensed from the syringe into transfer packs. The synthetic feeder cells were mixed and labeled with a transfer bag.

The total volume of feeder cell suspension in the transfer bag was calculated. A cell count sample was taken. A separate 3mL syringe was used for each sample and 4 x 1.0mL cell count samples were aspirated from the feeder cell suspension transfer bag using an optional injection port. Each sample was aliquoted into labeled frozen vials. Cell counting is performed, a multiplier is determined, a protocol is selected, and the multiplier is entered. The average value of the living cell concentration and the average value of the survival rate of the cells subjected to cell counting were measured. The upper and lower limits of the count were measured and confirmed to be within the limits.

The volume of feeder cell suspension was adjusted. The adjusted volume of feeder cell suspension after removal of the cell count sample is calculated. The total number of viable feeder cells was calculated. Additional feeder cells were obtained as needed. Additional feeder cells were thawed as needed. The 4 th feeder cell bag was placed in a zipper pack, thawed in a 37.0.+ -. 2.0 ℃ water bath or cytotherm min, and additional feeder cells were pooled. The volume was measured. The volume of feeder cells in the syringe was measured and recorded below (B). The new total volume of feeder cells was calculated. Feeder cells were added to the transfer bag.

Dilutions were prepared as needed and 4.5mL AIM-V medium was added to four 15mL conical tubes. Cell counts were prepared. A separate 3mL syringe was used for each sample, and 4 x 1.0mL cell count samples were removed from the feeder cell suspension transfer bag using an optional injection port. Cell counting and calculation were performed. The average viable cell concentration was determined from all four counts performed. The volume of the feeder cell suspension was adjusted and the adjusted volume of feeder cell suspension after removal of the cell count sample was calculated. Total feeder volume minus 4.0mL removed. The volume of feeder cell suspension required to obtain 5x10 ⁹ live feeder cells was calculated. Excess feeder cell volume was calculated. The volume of excess feeder cells to be removed was calculated. Excess feeder cells were removed.

Using a 1.0mL syringe and 16G needle, 0.15mL OKT3 is aspirated and OKT3 is added. The feeder cell suspension transfer bag was heat sealed.

Day 11-G-REX fill and inoculate set G-REX-500MCS. The "complete CM2 day 11 medium" was removed from the incubator and the medium was pumped into the G-REX500MCS. 4.5L of medium was pumped into G-REX500MCS and filled to the indicated line on the flask. Heat sealing and incubating the flask as needed. Feeder cell suspension transfer packs were welded to G-REX500MCS. Feeder cells were added to G-REX500MCS. And (5) heat sealing. The TIL suspension transfer pack was welded to a flask. TIL was added to G-REX500MCS. And (5) heat sealing. The G-REX500MCS is incubated at 37.0+ -2.0deg.C, CO2 percentage: 5.0+ -1.5% CO2.

And calculating the heat preservation range. Calculations were performed to determine the appropriate time to withdraw the G-REX500MCS from the incubator on day 16. Lower limit: incubation time +108 hours. Upper limit: incubation time +132 hours.

Excess TIL was stored frozen on day 11. The method is applicable to: excess TIL vials were frozen. Confirming that CRF had been set prior to freezing. Freezing and preserving. Vials were transferred from the rate controlled freezer to the appropriate storage. After the freezing is completed, the vials are transferred from the CRF to the appropriate storage container. The vials were transferred to appropriate storage. The storage location in LN2 is recorded.

Day 16-Medium preparation Pre-warmed AIM-V medium. The time to warm up the medium was calculated for medium bags 1, 2 and 3. Ensure that all bags have warmed for a duration of 12 to 24 hours. 10L Labtainer for the supernatant was set. The larger diameter end of the fluid pump transfer device was attached to one female port of the 10L Labtainer bag using a luer fitting. 10L Labtainer for the supernatant was set and labeled. 10L Labtainer for the supernatant was set. Ensure that all clips are closed before removal from the BSC. Note that: the supernatant bag is used during the TIL collection, which may be performed in parallel with the media preparation.

Thawing IL-2. 5X 1.1mL aliquots of IL-2 (6X 10 ⁶ IU/mL) (BR 71424) were thawed per bag of CTS AIM V medium until all ice was thawed. 100.0mL of GlutaMax ^TM was aliquoted. IL-2 was added to GlutaMax ^TM. Prepare CTS AIM V media bag for formulation. Prepare CTS AIM V media bag for formulation. Multistage Baxa pump. The preparation medium was prepared. GlutaMax ^TM +IL-2 was pumped into the bag. Monitoring parameters: temperature LED display: 37.0+/-2.0 ℃ and CO ₂ percent: 5.0+ -1.5% CO ₂. The complete CM4 day 16 medium was warmed. Preparing a diluent.

REP split bottles on day 16. Monitoring incubator parameters: temperature LED display: 37.0+/-2.0 ℃ and CO ₂ percent: 5.0.+ -. 1.5% CO ₂. The G-REX500MCS was removed from the incubator. A 1L transfer bag, labeled TIL suspension, was prepared and 1L weighed.

The volume of the G-REX-500MCS is reduced. About 4.5L of culture supernatant was transferred from G-REX-500MCS to 10LLabtainer.

The flask was prepared for TIL collection. After removal of the supernatant, all clamps to the red line were closed.

TIL collection is started. The flask was vigorously knocked and the medium was swirled to detach the cells, ensuring that all cells were detached.

TIL collection. All clamps leading to the TIL suspension transfer bag were released. Using GatheREX, the cell suspension was transferred to a TIL suspension transfer bag. Note that: it was ensured that the edge tilt was maintained until all cells and medium were collected. Adherent cells on the membrane were examined. The flask membrane was rinsed. The clip on the G-REX500MCS is closed. The transfer bag containing the TIL was heat sealed. Heat sealing 10L Labtainer containing supernatant. The weight of the transfer bag containing the cell suspension was recorded and the suspension volume calculated. A transfer pack for sample removal is prepared. Test samples were taken from the cell supernatants.

Sterility and BacT test samples. 1.0mL of the sample was removed from the prepared 15mL cone-labeled BacT. A cell count sample was taken. In the BSC, 4X 1.0mL of cell count samples were taken from the "TIL suspension" transfer packet using a separate 3mL syringe for each sample.

And taking out the mycoplasma sample. Using a 3mL syringe, 1.0mL was removed from the TIL suspension transfer bag and placed in a prepared 15mL conical tube labeled "mycoplasma diluent".

A transfer pack is prepared for inoculation. The TIL was placed in an incubator. Cell suspensions were removed from the BSC and placed in an incubator before required. Cell counting and calculation were performed. The cell count samples were first diluted by adding 0.5mL of the cell suspension to the prepared 4.5mL AIM-V medium to give a dilution of 1:10. The average value of the living cell concentration and the average value of the survival rate of the cells subjected to cell counting were measured. The upper and lower limits of the count are determined. Note that: dilution can be adjusted according to the desired cell concentration. The average viable cell concentration was determined from all four counts performed. The volume of the TIL suspension was adjusted. The adjusted volume of TIL suspension after removal of the cell count sample was calculated. Total TIL cell volume minus 5.0mL removed for testing.

The total number of viable TIL cells was calculated. The total number of flasks to be inoculated was counted. Note that: the maximum number of G-REX-500MCS flasks to be inoculated was five. If the calculated number of flasks to be inoculated exceeds five, only five flasks are inoculated with all available volumes of cell suspension.

The number of flasks used for subculture was counted. The number of bags of medium required in addition to the prepared bags was counted. One 10L "day 16 media of CM4" bag was prepared per two G-REX-500M flasks as calculated. The first GREX-500M flask was continued to be inoculated while additional medium was prepared and allowed to warm. The determined calculated number of other medium bags are prepared and warmed. G-REX500MCS is filled. Prepare to pump the medium and pump 4.5L of medium into G-REX500MCS. And (5) heat sealing. And (5) repeating filling. The flask was incubated. The target volume of TIL suspension to be added to the new G-REX500MCS flask was calculated. If the number of flasks calculated exceeds five, only five flasks are inoculated with all volumes of the cell suspension. The flask was prepared for inoculation. The G-REX500MCS was removed from the incubator. The G-REX500MCS is prepared for pumping. All clamps are closed except for the larger filter line. The TIL was removed from the incubator. Cell suspensions were prepared for inoculation. The "TIL suspension" transfer ladle was aseptically welded (according to process notes 5.11) to the pump inlet line. The TIL suspension bag was placed on the scale.

The flasks were inoculated with the TIL suspension. The calculated volume of TIL suspension was pumped into a flask. And (5) heat sealing. Fill the remaining flask.

The incubator was monitored. Incubator parameters: temperature LED display: 37.0+/-2.0 ℃; CO ₂ percent: 5.0+ -1.5% CO ₂. The flask was incubated.

The time frame for G-REX500MCS to be removed from the incubator on day 22 was measured.

Wash buffer preparation on day 22. A 10L Labtainer bag was prepared. In the BSC, a4 "plasma transfer device was attached to a 10L Labtainer bag by a luer fitting. A 10L Labtainer bag was prepared. Before transferring out of the BSC, all clips are closed. Note that: one 10L Labtainer bags were prepared for every two G-REX500MCS flasks to be collected. Plasmalyte was pumped into a 3000mL bag and air was removed from the 3000mL Origen bag by flipping the pump and manipulating the position of the bag. 25% human albumin was added to a 3000mL bag. 25% of human albumin was obtained with a final volume of 120.0 mL.

IL-2 dilutions were prepared. Using a 10mL syringe, 5.0mL of LOVO wash buffer was removed using a needleless injection port on the LOVO wash buffer bag. The LOVO wash buffer was dispensed into 50mL conical tubes.

Aliquots of CRF blank bag LOVO wash buffer. Using a 100mL syringe, 70.0mL of LOVO wash buffer was aspirated from the needleless injection port.

A1.1 mL aliquot of IL-2 (6X 10 ⁶ IU/mL) was thawed until all ice had melted. mu.L of IL-2 stock (6X 10 ⁶ IU/mL) was added to a 50mL conical tube labeled "IL-2 dilution".

Freezing and preserving preparation. 5 freezer boxes were placed at 2 to 8 ℃ to pre-treat them for the final product to be stored frozen.

Cell count dilutions were prepared. In BSC, 4.5mL AIM-V medium labeled "for cell count dilution" and lot number was added to 4 15mL conical tubes. Cell counts were prepared. The 4 frozen vials were labeled with vial numbers (1 to 4). The vials are saved in the BSC for use.

TIL collection on day 22. The incubator was monitored. Incubator parameters: temperature LED display: 37 plus or minus 2.0 ℃, CO2 percentage: 5% ± 1.5%. The G-REX500MCS flask was removed from the incubator. The TIL collection bag was prepared and labeled. Closing the additional joint. Volume reduction: about 4.5L of supernatant was transferred from G-REX500MCS to the supernatant bag.

The flask was prepared for TIL collection. TIL collection is started. The flask was vigorously knocked and the medium swirled to strip the cells. Ensure that all cells have exfoliated. TIL collection is started. All clamps to the TIL suspension collection bag were released. TIL collection. Using GatheRex, the TIL suspension was transferred to a 3000mL collection bag. Adherent cells on the membrane were examined. The flask membrane was rinsed. Closing the clips on the G-REX500MCS ensures that all clips are closed. The cell suspension was transferred to a LOVO source bag. Closing all the clamps. And (5) heat sealing. A4X 1.0mL cell count sample was taken.

Cell counting was performed. Cell counts and calculations were performed using NC-200 and procedure annotation 5.14. The cell count samples were first diluted by adding 0.5mL of the cell suspension to the prepared 4.5mL AIM-V medium. A dilution of 1:10 was obtained. The average viability, viable cell concentration, and total nuclear cell concentration of cells subjected to cell counting were determined. The upper and lower limits of the count are determined. The average viability, viable cell concentration, and total nuclear cell concentration of cells subjected to cell counting were determined. Weigh the LOVO source bag. The total number of viable TIL cells was calculated. The total number of nucleated cells was calculated.

Preparing mycoplasma diluent. 10.0mL was removed from one of the supernatant bags through the luer port and placed in a 15mL conical tube.

A "TIL G-REX collection" protocol was performed and the final product target volume was determined. The disposable kit is loaded. And taking out the filtrate bag. The filtrate volume was input. The filtrate vessel was placed on a laboratory bench. Plasmalyte was attached. It was confirmed that PlasmaLyte was attached and it was observed that PlasmaLyte was moving. Attaching the source container to the conduit, confirming that the source container has been attached. Confirm that PlasmaLyte is moving.

And finally preparing and filling. Target volume/bag calculation. The volumes of CS-10 and LOVO wash buffer to be formulated in the blank bag were calculated. Prepare a CRF blank bag.

The volume of IL-2 to be added to the final product was calculated. Desired final IL-2 concentration (IU/mL): 300IU/mL. IL-2 working reserve: 6X 10 ⁴ IU/mL. And assembling the connecting device. The 4S-4M60 was aseptically welded to the CC2 cell adaptor. The CS750 freezer bag was aseptically welded to the prepared wire bundle. The CS-10 bag was welded to the tip of 4S-4M 60. TIL was prepared using IL-2. Using a syringe of appropriate size, the measured amount of IL-2 was removed from an "IL-2 6X 10 ⁴" aliquot. The TIL bags were labeled. The formulated TIL bag was added to the apparatus. CS10 was added. The syringe is switched. About 10mL of air was drawn into the 100mL syringe and replaced on the device with the 60mL syringe. CS10 was added. Prepare CS-750 bag. Cells were dispensed.

Air was removed from the final product bag and a retentate was obtained. Once the last end product bag has been filled, all clamps are closed. 10mL of air was drawn into a new 100mL syringe and the syringe on the device was replaced. The retentate was dispensed into 50mL conical tubes and the tubes were labeled "retentate" and lot number. The air removal step is repeated for each bag.

The final product is ready for cryopreservation, including visual inspection. The freezer bag is stored on a temperature-reduced bag or at 2 to 8 ℃ prior to cryopreservation.

A cell count sample was taken. Using a pipette of appropriate size, 2.0mL of the retentate was removed and placed in a 15mL conical tube for cell counting. Cell counting and calculation were performed. Note that: only one sample was diluted to the appropriate dilution to verify that the dilution was sufficient. Additional samples were diluted to the appropriate dilution factor and counting continued. The average value of the living cell concentration and the average value of the survival rate of the cells subjected to cell counting were measured. The upper and lower limits of the count are determined. Note that: the dilution can be adjusted according to the desired cell concentration. The mean viable cell concentration and the mean viability were determined. The upper and lower limits of the count are determined. IFN-gamma was calculated. Heat sealing the final product bag.

The samples are marked and collected according to the following exemplary sampling plan.

Table 48: sample planning.

Sample of	Number of containers	Sample volume per addition	Container type
				* Mycoplasma species	1	1.0mL	15ML conical tube
Endotoxin (endotoxin)	2	1.0mL	2ML frozen vials
				Gram staining	1	1.0mL	2ML frozen vials
IFN-γ	1	1.0mL	2ML frozen vials
				Flow cytometry	1	1.0mL	2ML frozen vials
* Bac-T sterility	2	1.0mL	Bac-T bottle
				QC retentate	4	1.0mL	2ML frozen vials
Satellite vial	10	0.5mL	2ML frozen vials

Sterility and BacT tests. And (5) testing and sampling. In the BSC, 1.0mL samples were taken from the remaining cell suspension collected using a syringe of appropriate size and inoculated into an anaerobic jar. The above operation was repeated for the aerobic flask.

The final product is cryopreserved ready for rate controlled freezer (CRF). Confirm that CRF has been set. Setting CRF probes. The final product and samples were placed in CRF. The time required to reach 4 ℃ ± 1.5 ℃ was measured and CRF operation was continued. The CRF is completed and stored. After the operation is completed, the CRF is stopped. The cassette and vial were removed from the CRF. The cassettes and vials were transferred to gas phase LN2 for storage. The storage location is recorded.

The processing and analysis of the final drug product included the following tests: (day 22) cd3+ cells on day 22 REP were determined by flow cytometry; (day 22) gram stain method (GMP); bacterial endotoxin testing by gel clot LAL assay (GMP) (day 22); (day 16) BacT sterility analysis (GMP); (day 16) TD-PCR (GMP) detection of Mycoplasma DNA; acceptable appearance characteristics; (day 22) BacT sterile analysis (GMP) (day 22); (day 22) IFN-gamma analysis. Other efficacy assays as described herein are also used to analyze the TIL product.

Example 8: exemplary embodiment of GEN 3 amplification platform

Day 0

Tumor wash media were prepared. The medium is heated before starting. 5mL of gentamicin (50 mg/mL) was added to a 500mL HBSS bottle. 5mL of tumor wash medium was added to a 15mL Erlenmeyer flask for OKT3 dilution. Feeder cell bags were prepared. Feeder cells were aseptically transferred to feeder cell bags and stored at 37 ℃ until use or freezing. Feeder cells were counted if at 37 ℃. If frozen, the cells are thawed and then counted.

The optimum range of feeder cell concentrations is between 5X 10 ⁴ and 5X 10 ⁶ cells/mL. Four conical tubes with 4.5mL AIM-V were prepared. 0.5mL of cell fraction was added for each cell count. If the total number of living feeder cells is not less than 1X 10 ⁹ cells, the feeder cell concentration is adjusted. The volume of feeder cells removed from the first feeder cell bag was calculated so that 1 x 10 ⁹ cells were added to the second feeder cell bag.

Using a p1000 micropipette, 900 μl of tumor wash medium was transferred into OKT3 aliquots (100 μl). Using syringe and sterile technique, 0.6mL OKT3 is withdrawn and added to the second feeder cell bag. The culture medium volume was adjusted to a total volume of 2L. The second feeder cell bag was transferred to an incubator.

OKT3 formulation details: OKT3 may be aliquoted and frozen in a 100. Mu.L aliquot in a vial (1 mg/mL) of original stock concentration. Each 1mL vial was approximately 10X aliquots. Stored at-80 ℃. Day 0: 15. Mu.g/flask, i.e., 30ng/mL in 500 mL-up to about 60. Mu.L to 1 aliquot.

5ML of tumor wash medium was added to all wells of the 6-well plate labeled as redundant tumor fragments. Tumor wash medium was saved for further use in retaining tumor moisture during segmentation. 50mL of tumor wash medium was added to each 100mm Petri dish.

Tumors were sectioned into approximately 27mm ³ pieces (3X 3 mm) under the dissecting dish cover with a ruler as a reference. The middle fragment was dissected until 60 fragments were reached. The total number of final splits was counted and G-REX100MCS flasks (typically 60 splits/flask) were prepared based on the number of final splits produced.

The advantageous tissue fragments remain in the conical tubes marked as fragment tubes 1 to 4. The number of G-REX100MCS flasks inoculated with feeder cell suspension was calculated based on the number of initiated debris tubes.

The feeder cell bag was removed from the incubator and inoculated with G-REX100MCS. Marked D0 (day 0).

Tumor fragments were added to the culture in G-REX100 MCS. Under sterile conditions, the G-REX100MCS labeled with tumor fragment culture (D0) 1 and the cap of the 50mL conical tube labeled with fragment tube were unscrewed. The opened chip tube 1 is rotated while slightly lifting the lid of the G-REX100 MCS. The medium was added to the G-REX100MCS with the chips while rotating. The number of fragments transferred into the G-REX100MCS is recorded.

Once the fragments were at the bottom of the G-REX flask, 7mL of medium was aspirated, creating seven 1mL aliquots, 5mL for amplification characterization, and 2mL for sterile samples. 5 aliquots (final debris culture supernatant) for amplification characterization were stored at-20℃until needed.

One anaerobic BacT/Alert flask and one aerobic BacT/Alert flask were inoculated with 1mL of the final debris culture supernatant, respectively. The flasks were sampled and repeated.

On days 7-8

Feeder cell bags were prepared. In the case of freezing, the feeder cell bag was thawed in a 37℃water bath for 3-5 minutes. If frozen, feeder cells were counted. The optimum range of feeder cell concentrations is between 5X 10 ⁴ and 5X 10 ⁶ cells/mL. Four conical tubes with 4.5mL AIM-V were prepared. Cell fractions of 0.5mL per cell count were added to new frozen vial. The samples were thoroughly mixed and cell counted.

If the total number of viable feeder cells is ≡2×10 ⁹ cells, the next step is performed to adjust feeder cell concentration. The volume of feeder cells removed from the first feeder cell bag was calculated so that 2 x 10 ⁹ cells were added to the second feeder cell bag.

Using a p1000 micropipette, 900. Mu.L of HBSS was transferred to a 100. Mu.L OKT3 aliquot. Mixing was performed by pipetting up and down 3 times. Two aliquots were prepared.

OKT3 formulation details: OKT3 may be aliquoted and frozen in 100. Mu.L aliquots at the original stock solution concentration (1 mg/mL) from the vial. Each 1mL vial was about 10 x aliquots. Stored at-80 ℃. Day 7/8: 30. Mu.g/flask, i.e., 60ng/mL in 500 mL-up to 120. Mu.l-2 aliquots.

Using syringe and sterile technique, 0.6mL OKT3 is withdrawn and added to the feeder cell bag, ensuring complete addition. The culture medium volume was adjusted to a total volume of 2L. The second OKT3 aliquot was repeated and added to the feeder cell bag. The second feeder cell bag was transferred to an incubator.

G-REX100MCS flasks with feeder cell suspensions were prepared. The number of G-REX100MCS flasks to be treated was recorded according to the number of G-REX flasks produced on day 0. The G-REX flask was removed from the incubator and the second feeder cell bag was removed from the incubator.

The supernatant was removed prior to addition of the feeder cell suspension. A10 mL syringe was connected to the G-REX100 flask and 5mL of medium was withdrawn. Five 1mL aliquots were created, 5mL for the amplification characterization and 5 aliquots for the amplification characterization (final debris culture supernatant) were stored at-20 ℃ until the requirements of the sponsor were made. Each G-REX100 flask was labeled and repeated.

5-20X 1mL samples were used for characterization, depending on the number of flasks:

● 5mL = 1 culture flask

● 10ML = 2 culture flasks

● 15ML = 3 culture flasks

● 20ML = 4 flasks

Feeder cells were continuously seeded into G-REX100MCS and the flasks were repeated for each G-REX100 MCS. Using a sterile transfer method, 500mL of a second feeder cell bag was transferred by gravity into each G-REX100MCS flask by weight (assuming 1 g=1 mL), and the transfer was recorded. Cultures labeled on day 7 were replicated for each G-REX100 bottle. The G-REX100MCS flask was transferred to an incubator.

Day 10-11

The first G-REX100MCS flask was removed and 7mL of the pretreatment culture supernatant was removed under sterile conditions using a 10mL syringe. Seven 1mL aliquots were created, 5mL for amplification characterization and 2mL for sterile samples.

The flask was carefully mixed and 10mL of supernatant was removed using a new 10mL syringe and transferred to a 15mL tube labeled D10/11 mycoplasma supernatant.

Carefully mix the flasks, take the following volumes using a new syringe, depending on the number of flasks to be treated:

● 1 culture flask = 40mL

● 2 Flasks = 20 mL/flask

● 3 Flasks = 13.3 mL/flask

● 4 Flasks = 10 mL/flask

A total of 40mL should be aspirated from all flasks, pooled in a 50mL conical tube labeled "day 10/11 QC sample" and stored in an incubator until needed. Cell counting was performed and cells were distributed.

5 Aliquots (pretreatment culture supernatants) for amplification characterization were stored at-20℃until needed. One anaerobic BacT/Alert flask and one aerobic BacT/Alert flask were inoculated with 1mL of pretreatment culture supernatant, respectively.

The cell suspension was continued to be transferred to G-REX-500MCS and repeated for each G-REX100 MCS. Using aseptic conditions, the contents of each G-REX100MCS were transferred to the G-REX-500MCS, with about 100mL of liquid transfer monitored at a time. When the volume of the G-REX100MCS was reduced to 500mL, the transfer was stopped.

During the transfer step, 10mL of the cell suspension was aspirated into the syringe using a 10mL syringe and from the G-REX100 MCS. The operations were performed according to the instructions according to the number of flasks. If only 1 flask: a total of 20mL was withdrawn using two syringes. If 2 culture flasks: 10mL of each flask was removed. If 3 culture flasks: 7mL of each flask was removed. If 4 culture flasks: 5mL of each flask was removed. The cell suspension was transferred to a conventional 50mL conical tube. Kept in incubator until the cell counting step and QC samples. The total number of cells required for QC is about 20e6 cells: 4X 0.5mL cell count (cell count was first undiluted).

The amount of cells required for analysis is as follows:

1. at least 10X 10 ⁶ cells are used for efficacy assays, such as those described herein, or for IFN-gamma or granzyme B assays

2.1X10 ⁶ cells for Mycoplasma

3.5X10 ⁶ cells for CD3+/CD45+ flow cytometry

The G-REX-500MCS flask was transferred to an incubator.

QC samples were prepared. In this example, at least 15×10 ⁸ cells are required for analysis. The analysis includes: cell count and viability; mycoplasma (1X 10 ⁶ cells/mean survival concentration) flow cytometry (5X 10 ⁶ cells/mean survival concentration; 5X 10 ⁶ cells-1X 10 ⁶ cells; 8-10X 10 ⁶ cells were required for IFN-gamma analysis).

The volume of the cell fraction cryopreserved at 10×10 ⁶ cells/mL was calculated and the number of vials to be prepared was calculated.

Day 16-17

Washing buffer preparation (1% HSA PLASMALYTE A). HSA and Plasmalyte were transferred to a 5L bag to prepare a LOVO wash buffer. A total volume of 125mL of 25% HSA was transferred to a 5L bag using sterile conditions. 10mL or 40mL of wash buffer was removed and transferred to an "IL-2 6X 10 ⁴ IU/mL tube" (10 mL if IL-2 was prepared in advance, or 40mL if IL-2 was freshly prepared).

The volume of reconstituted IL-2 added to plasmalyte+1% HSA was calculated: the volume of restored IL-2= (final concentration of IL-2 x final volume)/specific activity of IL-2 (based on standard analysis). The final concentration of IL-2 was 6X 10 ⁴ IU/mL. The final volume was 40mL.

The calculated initial volume of IL-2 required to restore IL-2 was removed and transferred to an "IL-2 6X 10 ⁴ IU/mL" tube. 100. Mu.L of IL-2 6 X10: 10 ⁶ IU/mL from a pre-prepared aliquot was added to a labeled "IL-2 6X 10 ⁴ IU/mL" tube containing 10mL of LOVO wash buffer.

About 4500mL of supernatant was removed from the G-REX-500MCS flask. The remaining supernatant was spun and the cells transferred to a cell collection bag. All G-REX-500MCS flasks were repeated.

60ML of supernatant was removed and added to the supernatant tube for quality control analysis, including mycoplasma detection. Stored at +2-8deg.C.

And (5) collecting cells. Cells were counted. Four 15mL conical tubes with 4.5mL AIM-V were prepared. These conical tubes may be prepared in advance. Optimal range = between 5 x 10 ⁴ and 5 x 10 ⁶ cells/mL. (1:10 dilution recommended). 500 μl CF was added to 4500 μl AIM V prepared previously for a 1:10 dilution. The dilution factor is recorded.

Calculate TC (total cells) =average total cell concentration (pre-LOVO TC concentration) (survival+death) of pre-LOVO (survival+death) X source bag volume

Calculate TVC (total living cells) =average total living cell concentration (pre-LOVO TVC) (living) XLOVO source bag volume of pre-LOVO (living)

When the total number of cells (TC) was >5×10 ⁹, 5×10 ⁸ cells were removed for cryopreservation as MDA-reserved samples. 5×10 ⁸ +.average TC concentration (step 14.44) =volume to be removed.

When the total number of cells (TC) was 5×10 ⁹ or less, 4×10 ⁶ cells were removed for cryopreservation as MDA-reserved samples. 4×10 ⁶ = average TC concentration = volume to be removed.

When determining the total cell number, the number of cells to be removed should allow for the retention of 150X 10 ⁹ living cells. The TVC of the pre-LOVO was confirmed to be 5 x10 ⁸ or 4 x10 ⁶ or not applicable. The volume of cells to be removed is calculated.

The remaining total cells remaining in the bag were calculated. TC (total cells) of pre-LOVO was calculated. [ average Total cell concentration Xresidual volume=Pre-LOVO residual TC ]

The corresponding procedure in table 49 was selected based on the total number of cells remaining.

Table 49: total number of cells.

Total cells:	Retentate (mL)
		0< Total cells ∈31X10- ⁹	115
31×10 ⁹ < Total cells ∈71×10- ⁹	165
		71×10 ⁹ < 110×10 Total cells- ⁹	215
110×10 ⁹ < Total cells ∈115×10- ⁹	265

The volume of IL-2 addition corresponding to the procedure used was selected. The volume calculation is as follows: retentate volume x2 x 300IU/mL = IU of desired IL-2. IU/6×10 ⁴ IU/mL of desired IL-2 = volume of IL-2 added after the LOVO bag. Record all added volumes. Samples were obtained in frozen vials for further analysis.

The cell products were thoroughly mixed. All bags are sealed for further processing, including cryopreservation, as appropriate.

The frozen vial samples obtained were subjected to endotoxin, IFN-gamma, sterility and other analyses as required.

Example 9: GEN 2 and GEN 3 exemplary procedure

This example shows the Gen 2 and Gen 3 processes. The processes Gen 2 and Gen 3TIL are typically composed of autologous TIL derived from the individual patient (tumor resected by surgery) and then amplified ex vivo. The initial first expansion step of the Gen 3 process is cell culture in the presence of interleukin-2 (IL-2) and monoclonal antibody OKT3, which monoclonal antibody OKT3 targets the T cell co-receptor CD3 on the scaffolds of irradiated Peripheral Blood Mononuclear Cells (PBMCs).

The production of Gen 2TIL product consists of two stages: 1) Pre-rapid amplification (pre-REP), and 2) rapid amplification protocol (REP). During pre-REP, resected tumors were dissected into < 50 fragments of 2-3mm each dimension, which were incubated with serum-containing medium (supplemented with 10% HuSAB of RPMI 1640 medium) and 6,000IU/mL interleukin-2 (IL-2) for a period of 11 days. On day 11, TIL was collected and introduced into large-scale secondary REP amplification. REP consists of: in a 5L volume of CM2 supplemented with 3000IU/mL rhIL-2, 200X 10 ⁶ live cells from pre-REP were activated for 5 days in a co-culture of 5X 10 ⁹ irradiated allogeneic PBMC feeder cells loaded with 150. Mu.g of monoclonal anti-CD 3 antibody (OKT 3). On day 16, the culture volume was reduced by 90% and the cell fraction was split into multiple G-REX-500 flasks, 1X 10 ⁹ live lymphocytes/flask, supplemented with CM4 to 5L. TIL was further cultured for 6 days. REP was collected on day 22, washed, formulated and cryopreserved, and then transported to the clinical site for infusion at-150 ℃.

The production of Gen 3TIL product consists of three stages: 1) initiating a first amplification protocol, 2) a rapid second amplification protocol (also known as rapid amplification stage or REP), and 3) subculture split. To achieve initial first amplified TIL proliferation, resected tumors were cut into 120 pieces of 2-3mm each dimension. On day 0 of initiation of the first expansion, a feeder layer of about 2.5X10- ⁸ allograft irradiated PBMC feeder cells loaded with OKT-3 was established on a surface area of about 100cm ² in each of the 3 100MCS containers. Tumor fragments were distributed in 3 100MCS containers, each of which was incubated with 500mL of serum-containing CM1 medium and 6,000IU/mL interleukin-2 (IL-2) and 15 μg OKT-3 for a period of 7 days. On day 7, REP was initiated by: about 5X10 ⁸ additional feeder cell layers of OKT-3 loaded, allograft-irradiated PBMC feeder cells were incorporated into the tumor disruption culture stage in each of three 100MCS vessels, cultured with 500mL CM2 medium and 6,000IU/mL IL-2 and 30 μ gOKT-3 by transferring OKT3 loaded feeder cells into the 100MCS vessel using a closed system fluid, activating the entire initial first expansion culture in the same vessel to enhance REP initiation. For Gen 3, til longitudinal expansion or split involves a treatment step in which the whole cell culture is fluid transferred through a closed system, scaled to a larger vessel, transferred (from 100M flask to 500M flask), and an additional 4l of CM4 medium is added. REP was collected on day 16, washed, formulated and cryopreserved, and then shipped to a clinical site at-150 ℃ for infusion.

Overall, the Gen 3 process is a shorter, more scalable and easily modifiable amplification platform that will accommodate robust manufacturing and process comparability.

Table 50: comparison of exemplary Gen 2 and exemplary Gen 3 manufacturing processes.

On day 0, tumors were washed 3 times for both procedures, randomly grouping the fragments into two pools; one pool per process. For the Gen 2 process, the fragments were transferred to a GREX MCS flask with 1L CM1 medium containing 6,000IU/mL rhIL-2. For the Gen 3 procedure, the fragments were transferred to a G-REX100MCS flask with 500mL CM1 containing 6,000IU/mL rhIL-2, 15 μg OKT-3, and 2.5X10 ⁸ feeder cells. Inoculation of TIL on Rep-initiated days was performed on different days according to the respective procedure. For the Gen 2 procedure, the volume of the G-REX100MCS flask was reduced by 90%, and the collected cell suspension was transferred to a new G-REX-500MCS to initiate REP priming on day 11 in CM2 medium containing IL-2 (3000 IU/mL) plus 5X 10 ⁹ feeder cells and OKT-3 (30 ng/mL). Cells in each protocol were expanded and split on day 16 into multiple G-REX-500MCS flasks with CM4 medium and IL-2 (3000 IU/mL). Cultures were then collected and cryopreserved on day 22 in each protocol. For the Gen 3 process, REP initiation was performed on day 7, where REP initiation was performed using the same G-REX100 MCS. Briefly, 500mL of CM2 medium containing IL-2 (6000 IU/mL), 5X 10 ⁸ feeder cells and 30. Mu.g OKT-3 was added to each flask. On days 9-11, the cultures were scaled up longitudinally. The entire volume of G-REX100M (1L) was transferred to G-REX-500MCS, and 4L of CM4 containing IL-2 (3000 IU/mL) was added. The flask was incubated for 5 days. Cultures were collected on day 16 and cryopreserved.

Three different tumors, two lung tumors (L4054 and L4055) and one melanoma (M1085T) were included in the comparison.

For L4054 and L4055, CM1 (medium 1), CM2 (medium 2) and CM4 (medium 4) media were prepared in advance and maintained at 4 ℃. CM1 and CM2 media were prepared without filtration to compare cell growth with and without media filtration.

For L4055 tumors, the medium was warmed up to 24 hours at 37 ℃ at REP start-up and scale-up.

As a result. For the total viable cells achieved, the results for Gen 3 are within 30% of the results for Gen 2. After restimulation, the Gen 3 end product showed higher IFN- γ production. The Gen 3 end product showed increased clonal diversity as determined by the presence of the total unique CDR3 sequence. The Gen 3 end product showed a longer average telomere length.

Prep and REP amplification of Gen 2 and Gen 3 procedures followed the procedure described above. For each tumor, both pools contained an equal number of fragments. Due to the smaller size of the tumor, the maximum number of fragments per flask could not be achieved. The total pre-REP cells (TVCs) were collected and counted on day 11 of the Gen 2 process and day 7 of the Gen 3 process. To compare the two pre-REP groups, the cell count was divided by the number of fragments provided in the culture to calculate an average of the viable cells per fragment. As indicated in table 51 below, more cells were grown throughout the Gen 2 process on a per-fragment basis as compared to the Gen 3 process. The extrapolated calculation of the expected TVC number on day 11 of the Gen 3 process is calculated by dividing the prerep TVC by 7 and then multiplying by 11.

Table 51: pre-REP cell count

* L4055, unfiltered medium.

For the Gen 2 and Gen 3 processes, TVCs were counted according to the process conditions, producing a percentage of viable cells per day of the process. At the time of collection, day 22 (Gen 2) and day 16 (Gen 3) cells were collected and TVC counts were established. The TVC was then divided by the number of fragments provided on day 0to calculate the average of viable cells per fragment. The amplification factor was calculated by dividing the collected TVC by the REP-initiated TVC. As shown in table 52, fold amplification was similar for Gen 2 and Gen 3, L4054; in the case of L4055, the amplification factor of the Gen 2 process is higher. Specifically, in this case, the medium is warmed 24 before the REP start-up day. For M1085T, a higher amplification factor was also observed in Gen 3. The extrapolated calculation of the expected TVC number on day 22 of the Gen 3 process is calculated by dividing the REP TVC by 16 and then multiplying by 22.

Table 52: total viable cell count and fold expansion of the TIL final product.

* L4055, unfiltered medium.

Table 53: percent survival of TIL end product: at the time of collection, the final TIL REP product was compared to a release criterion for% survival. All conditions of the Gen2 and Gen 3 processes exceeded the 70% viability criteria, with comparability in terms of process and tumor.

After collection, the final TIL REP product was compared to a release criterion for% survival. All conditions of the Gen 2 and Gen 3 processes exceeded the 70% viability criterion, with comparability in terms of process and tumor.

Table 53: percent survival of REP (TIL end product)

Since the number of fragments per flask was below the maximum required number, the estimated cell count at the time of collection was calculated for each tumor. The evaluation is based on the following expectations: clinical tumors were large enough to vaccinate 2 or 3 flasks on day 0.

Table 54: estimated cell count calculations were extrapolated to full-size 2 and 3 flasks of the Gen 3 process.

Immunophenotyping-phenotypic marker comparison of TIL end products. Three tumors, L4054, L4055 and M1085T, all underwent TIL amplification during Gen 2 and Gen 3. After collection, the REP TIL end products were subjected to flow cytometry analysis to test for purity, differentiation and memory markers. The percentage of TCR a/b+ cells exceeded 90% for all conditions.

TIL collected from the Gen 3 process showed higher expression of CD8 and CD28 than TIL collected from the Gen 2 process. The Gen 2 process showed a higher percentage of cd4+.

TIL collected from Gen 3 procedure showed higher expression in central memory compared to TIL collected from Gen 2 procedure.

Activation and depletion markers were analyzed in TIL from both tumors L4054 and L4055 to compare the final TIL products from the Gen2 and Gen 3TIL amplification processes. The activation and depletion markers of Gen2 and Gen 3 processes are comparable.

Interferon gamma secretion upon restimulation. TIL was re-stimulated overnight for L4054 and L4055 using coated anti-CD 3 discs on the collection days, i.e., day 22 of Gen 2 and day 16 of Gen 3. M1085T was restimulated with anti-CD 3, CD28 and CD137 beads. Under all conditions, the supernatant was collected after 24 hours of restimulation and frozen. The supernatants from both processes were assessed for ifnγ analysis by ELISA using the same ELISA plate. Higher ifnγ production from the Gen 3 process was observed in the three tumors analyzed.

Measurement of IL-2 content in the medium. To compare IL-2 consumption between Gen 2 and Gen 3 processes, cell supernatants were collected on REP start, scale up and harvest days for tumors L4054 and L4055. The amount of IL-2 in cell culture supernatants was determined by Quantitate ELISA kit from R & D. General trends indicate that IL-2 concentration remains higher during Gen 3 when compared to Gen 2. This is probably due to the higher IL-2 concentration at REP start of Gen 3 (6000 IU/mL) and the residue of medium throughout.

Metabolic substrate and metabolite analysis. The content of metabolic substrates such as D-glucose and L-glutamine is determined as a surrogate for the overall medium consumption. The reciprocal metabolites thereof, such as lactic acid and ammonia, are assayed. Glucose is a monosaccharide in the medium that the granulosa body uses to generate energy in the form of ATP. When glucose is oxidized, lactic acid (lactate is an ester of lactic acid) is produced. Lactate is produced in large amounts during the exponential growth phase of cells. High levels of lactate can negatively impact the cell culture process.

Spent medium of L4054 and L4055 was collected at the REP start, longitudinal scale up and collection days of the Gen 2 and Gen 3 processes. Spent medium was collected on days 11, 16 and 22 of Gen 2 and spent medium was collected on days 7, 11 and 16 of Gen 3. The supernatant was analyzed for glucose, lactate, glutamine, glutaMax ^TM and ammonia concentrations using a CEDEX bioanalyzer.

L-glutamine is a labile essential amino acid required in cell culture media formulations. Glutamine contains an amine, and this amide structural group can transport and deliver nitrogen to cells. When L-glutamine oxidizes, cells produce toxic ammonia as a byproduct. To counteract the degradation of L-glutamine, the medium of the Gen 2 and Gen 3 process was supplemented with GlutaMax ^TM, which is more stable in aqueous solution and does not spontaneously degrade. In both tumor lines, the Gen 3 group showed a decrease in L-glutamine and GlutaMax ^TM during the course, as well as an increase in ammonia in the overall REP. In the Gen 2 group, constant L-glutamine and GlutaMax ^TM concentrations were observed, as well as a slight increase in ammonia production. For ammonia, the Gen 2 and Gen 3 processes were comparable at the time of collection, with slightly different L-glutamine degradation.

Telomeres were repeated by Flow-FISH. The average length of telomere repeats over L4054 and L4055 during Gen 2 and Gen 3 was determined using Flow-FISH techniques. The measurement of the Relevant Telomere Length (RTL) was calculated using the telomere PNA kit/FITC from DAKO for flow cytometry analysis. Gen 3 shows a telomere length comparable to Gen 2.

CD3 analysis. To determine the clonal diversity of the cellular products produced in each process, the collected TIL end products of L4054 and L4055 were sampled and analyzed for clonal diversity by sequencing of the CDR3 portion of the T cell receptor.

Table 55 shows a comparison of the percentage of unique CDR3 sequences shared between Gen 2 and Gen 3 on L4054 on TIL-collected cell products. Gen 3 shares 199 sequences with the Gen 2 end product, corresponding to 97.07% of the unique CDR3 sequences of the first 80% of Gen 2 shared with the Gen 3 end product.

Table 55: comparison of shared uCDR sequences between Gen 2 and Gen 3 Processes on L4054

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Table 56 shows a comparison of the percentage of unique CDR3 sequences shared on L4055 on TIL-harvested cell products between Gen 2 and Gen 3. Gen 3 shares 1833 sequences with the Gen 2 end product, 99.45% of the unique CDR3 sequences corresponding to the first 80% of the Gen 2 end product being shared with the Gen 3 end product.

Table 56: comparison of shared uCDR sequences between Gen 2 and Gen 3 Processes on L4055

CM1 and CM2 media were prepared in advance without filtration and maintained at 4 ℃ until used for tumor L4055 for Gen 2 and Gen 3 processes.

For L4055 tumors, on the REP start-up day, the medium was warmed at 37 ℃ for 24 hours for use in the Gen 2 and Gen3 processes.

LDH was not measured in the supernatant collected in the process.

M1085T TIL cell counts were performed using a K2 cellometer cell counter.

On tumor M1085T, the sample was not available, e.g., supernatant for metabolic analysis; TIL products for activation and depletion marker assays, telomere length and CD3-TCR vb assays.

Conclusion (d). This example compares the functional quality attributes of 3 independent donor tumor tissues, plus the characterization of the amplified phenotype and the media consumption between Gen 2 and Gen 3 processes.

Gen 2 and Gen 3 pre-REP and REP expansion comparisons were assessed based on the viability of the total living cells and total nucleated cell populations produced. The TVC cell dose at the time of harvest was not comparable between Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than Gen 2, about 40% of total viable cells collected at the time of collection.

Assuming pre-REP collection was performed on day 11 instead of day 7 and REP collection was performed on day 22 instead of day 16, extrapolated cell numbers for the Gen 3 process were calculated. In both cases, gen 3 showed a closer number on TVC than the Gen 2 process, indicating an early activation enhanced TIL increase.

In the case of extrapolated values of additional flasks (2 or 3) during Gen 3, the treated tumor size was assumed to be larger, reaching the maximum number of fragments required for each procedure as described. It was observed that the Gen 3 process could achieve similar doses on TVCs collected on day 16 as compared to the Gen 2 process on day 22. This observation is important, indicating that early activation of cultures reduces TIL treatment time.

Gen 2 and Gen 3 pre-REP and REP expansion comparisons were assessed based on the viability of the total living cells and total nucleated cell populations produced. The TVC cell dose at the time of harvest was not comparable between Gen 2 (22 days) and Gen 3 (16 days). The Gen 3 cell dose was lower than Gen 2, approximately 40% of the total viable cells collected at the time of collection.

For phenotypic characterization, higher cd8+ and cd28+ expression was observed on three tumors during Gen 3 compared to Gen 2 process.

The Gen 3 process showed a slightly higher central memory compartment compared to the Gen 2 process.

Despite the short duration of the Gen 3 process, the Gen 2 and Gen 3 processes showed comparable activation and depletion markers.

Among the three tumors analyzed, ifnγ production on Gen 3 end product was 3-fold higher than Gen 2. This data suggests that the Gen 3 process produces a powerful and more potent TIL product compared to the Gen 2 process, which may be attributed to higher expression of CD8 and CD28 on Gen 3. Phenotypic characterization showed a positive trend for cd8+, cd28+ expression of Gen 3 compared to the Gen 2 process on three tumors.

The telomere length of the TIL end products of Gen 2 and Gen 3 were comparable.

The glucose and lactate levels of the Gen 2 and Gen 3 end products were comparable, indicating that the nutrient levels on the medium of the Gen 3 process were not affected, as there was no decrement removal on each day of the process and the overall medium volume in the process was smaller compared to Gen 2.

The overall processing time of the Gen 3 process is reduced by about two times compared to the Gen 2 process, which will significantly reduce the commercial Cost (COG) of TIL products amplified by the Gen 3 process.

IL-2 consumption showed a general trend of IL-2 consumption during Gen 2, whereas IL-2 was higher during Gen 3, since the old medium was not removed.

Through CDR3 TCRab sequence analysis, the Gen 3 process showed higher clonal diversity.

The addition of feeder cells and OKT-3 on day 0 of prerep allowed early activation of TIL and allowed TIL growth using the Gen 3 process.

Table 57 describes various embodiments and results of the Gen 3 process as compared to the current Gen 2 process.

Table 57: exemplary Gen 3 Process characterization

Example 10: exemplary GEN 3 procedure (also referred to as GEN 3.1)

This example describes other studies on "comparability between Gen 2 and Gen 3 processes for TIL amplification". The Gen 3 process is modified to include an activation step early in the process, with the goal of increasing the final Total Viable Cell (TVC) output while maintaining the phenotype and functional profile. As described below, the Gen 3 embodiment is modified to another embodiment and is referred to herein in this example as Gen 3.1.

In some embodiments, the Gen 3.1TIL manufacturing process has four operator interventions:

1. tumor debris separation and activation: on day 0 of the procedure, tumors were dissected and final fragments (up to 240 total fragments) of approximately 3×3mm each were produced and cultured in 1 to 4G-REX 100MCS flasks. Each flask contained up to 60 pieces, 500mL of CM1 or DM1 medium, and was supplemented with 6,000IU rhIL-2, 15 μg OKT3, and 2.5X10 ⁸ irradiated allogeneic monocytes. Cultures were incubated at 37℃for 6 to 8 days.

Til culture reactivation: on days 7 to 8, in both cases, cultures were supplemented by slow addition of CM2 or DM1 medium supplemented with 6,000IU rhIL-2, 30. Mu.g OKT3 and 5X 10 ⁸ irradiated allogeneic monocytes. Care is taken not to interfere with the existing cells at the bottom of the bottle. The cultures were incubated at 37℃for 3 to 4 days.

3. Cultivation scale is longitudinally enlarged: carried out on days 10 to 11. During the longitudinal expansion of the culture scale, the entire contents of the G-REX100MCS were transferred in both cases to G-REX500MCS flasks containing 4L of CM4 or DM2 supplemented with 3,000IU/mL IL-2. The flasks were incubated at 37℃for 5 to 6 days until collection.

4. Collection/washing/formulation: on days 16 to 17, the flask volumes were reduced and pooled. The cells were concentrated and washed with PLASMALYTE APH 7.4.4 containing 1% has. The washed cell suspension was formulated with CryoStor10 at a 1:1 ratio, supplemented with rhIL-2 to a final concentration of 300IU/mL.

DP was cryopreserved by controlled rate freezing and stored in gas phase liquid nitrogen. * The complete standard TIL medium 1,2 or 4 (CM 1, CM2, CM 4) may be substituted for CTS ^TMOpTmizer^TM T cell serum-free expansion medium called defined medium (DM 1 or DM 2), as mentioned above.

Description of the process. On day 0, tumors were washed 3 times, and then broken into 3 x 3 final pieces. After the whole tumor was broken up, the final fragments were then equally randomized and divided into three pools. One pool of randomized chips was introduced into each group, and the same number of chips was added according to the three experimental matrices.

During the whole TIL amplification procedure, tumor L4063 was amplified using standard medium and tumor L4064 was amplified using defined medium (CTS OpTmizer). The components of the culture medium are described herein.

CM1 complete medium 1: RPMI+Glutamine supplemented with 2mM GlutaMax ^TM, 10% human AB serum, gentamicin (50. Mu.g/mL), 2-mercaptoethanol (55. Mu.M). The final medium formulation was supplemented with 6000IU/mL IL-2.

CM2 complete medium 2:50% CM1 medium+50% AIM-V medium. The final medium formulation was supplemented with 6000IU/mL IL-2.

CM4 complete medium 4: AIM-V supplemented with GlutaMax ^TM (2 mM). The final media formulation was supplemented with 3000IU/mL IL-2.

CTS OpTmizer CTS ^TMOpTmizer^TM T cell expansion basal medium supplemented with CTS ^TMOpTmizer^TM T cell expansion supplement (26 mL/L).

DM1: CTS ^TMOpTmizer^TM T cell expansion basal medium supplemented with CTS ^TMOpTmizer^TM T cell expansion supplement (26 mL/L), CTS ^TM immune cell SR (3%) and GlutaMax ^TM (2 mM). The final formulation was supplemented with 6,000IU/mL IL-2.

DM2: CTS ^TMOpTmizer^TM T cell expansion basal medium supplemented with CTS ^TMOpTmizer^TM T cell expansion supplement (26 mL/L), CTS ^TM immune cell SR (3%) and GlutaMax ^TM (2 mM). The final formulation was supplemented with 3,000IU/mL IL-2.

All types of media used, i.e. Complete (CM) and Defined (DM) media, were prepared in advance, kept at 4 ℃ until the day before use, and warmed up to 24 hours in advance at 37 ℃ before the treatment day in an incubator.

For both tumors, TIL culture reactivation was performed on day 7. Scaling up occurred on day 10 for L4063 and on day 11 for L4064. Both cultures were collected and cryopreserved on day 16.

The result achieved. Cell counts and percent viability for Gen 3.0 and Gen 3.1 processes were determined. Amplification under all conditions followed the details described in this example.

For each tumor, the fragments were divided into three equal number pools. Due to the smaller size of the tumor, the maximum number of fragments per flask could not be achieved. For three different procedures, total living cells and cell viability under each condition were assessed. Cell counts were determined as TVC for reactivation on day 7, TVC for scale up on day 10 (L4064) or day 11 (L4063), and TVC collected on day 16/17.

Cell counts on days 7 and 10/11 were considered FIO. The fold amplification was calculated by dividing the TVC at the time of collection on day 16/17 by the TVC on day 7 reactivation. To compare the three groups, the TVC on the harvest day was divided by the number of fragments added to the culture on day 0 to calculate the average of viable cells on a per-fragment basis.

Cell count and viability assays were performed on L4063 and L4064. On both tumors, the Gen3.1 test procedure produced more cells per fragment than the Gen 3.0 procedure.

Total viable cell count and fold expansion; percent survival during the procedure. After reactivation, longitudinal scale up and collection, percent survival under all conditions was obtained. After collection on day 16/17, the final TIL was compared against a release criterion for percent survival. All conditions exceeded the 70% viability criterion, with comparability in terms of course and tumor.

Immunophenotyping-phenotypic characterization of TIL end product. The final product was subjected to flow cytometry analysis to test for purity, differentiation and memory markers. The population percentages of TCR α/β, cd4+ and cd8+ cells were consistent under all conditions.

Amplification phenotyping of REP TIL was performed. TIL products showed a higher percentage of cd4+ cells in Gen 3.1 compared to Gen 3.0 in both tumors and a higher percentage of cd28+ cells from the cd8+ population in Gen 3.0 compared to Gen 3.1.

TIL collected from Gen 3.0 and Gen 3.1 processes showed phenotypic markers comparable to CD27 and CD56 expression on cd4+ and cd8+ cells, as well as comparable CD28 expression on cd4+ gated cell populations. Memory tag comparison of TIL end product:

frozen samples of TIL collected on day 16 were stained for analysis. The TIL memory state is comparable between gen3.0 and gen3.1 processes. Activation of TIL end product and depletion marker comparison:

Activation and depletion markers are comparable between Gen 3.0 and Gen 3.1 processes gated on cd4+ and cd8+ cells.

Interferon gamma secretion upon restimulation. The collected TILs were restimulated overnight using coated anti-CD 3 plates of L4063 and L4064. In both tumors analyzed, higher ifnγ production from the Gen 3.1 process was observed compared to the Gen 3.0 process.

Measurement of IL-2 content in the medium. To compare IL-2 consumption under all conditions and procedures, cell supernatants were collected and frozen at the beginning of reactivation on day 7, scale-up on day 10 (L4064)/day 11 (L4063), and collection on day 16/17. The supernatant was then thawed and analyzed. The amount of IL-2 in cell culture supernatants was determined by the manufacturer's protocol.

The overall process of Gen 3 and Gen 3.1 was comparable in terms of IL-2 consumption throughout the evaluation under the same medium conditions. IL-2 concentration (pg/mL) analysis of the collected used media of L4063 and L4064.

Metabolite analysis. For each condition, spent medium supernatants were collected from L4063 and L4064 at day 7 reactivation onset, day 10 (L4064)/day 11 (L4063) scale-up, and day 16/day 17 collection of L4063 and L4064. The supernatant was analyzed for glucose, lactate, glutamine, glutaMax ^TM and ammonia concentrations using a CEDEX bioanalyzer.

The glucose concentration of the defined medium was higher than that of the complete medium (2 g/L), 4.5g/L. Overall, the glucose concentration and consumption of the Gen 3.0 and Gen 3.1 processes were comparable in each media type.

An increase in lactate was observed and was comparable between Gen 3.0 and Gen 3.1 conditions and between the two media used for reactivation amplification (complete medium and defined medium).

In some cases, standard basal medium contains 2mM L-glutamine and is supplemented with 2mM GlutaMax ^TM to compensate for the natural degradation of L-glutamine to L-glutamic acid and ammonia under culture conditions.

In some cases, the defined (serum-free) medium used was free of L-glutamine compared to basal medium, supplemented with only dipeptide of the GlutaMax ^TM.GlutaMax^TM line L-alanine and L-glutamine at a final concentration of 2mM, was more stable than L-glutamine in aqueous solution, and did not spontaneously degrade to glutamic acid and ammonia. In contrast, dipeptides gradually break down into individual amino acids, thereby maintaining a low but sufficient concentration of L-glutamine to maintain stable cell growth.

In some cases, the concentrations of glutamine and GlutaMax ^TM decreased slightly on the scale-up day, but showed an increase on the harvest day to a similar or closer level than on the reactivation day. For L4064, during the entire process, glutamine and GlutaMax ^TM concentrations showed slight degradation at similar rates under different conditions.

The ammonia concentration was higher in samples grown in standard medium containing 2mM glutamine+2 mM GlutaMax ^TM compared to samples grown in defined medium containing 2mM GlutaMax ^TM. Furthermore, as expected, there is a gradual increase or accumulation of ammonia during the culture. There were no differences in ammonia concentration at the three different test conditions.

Repeated telomeres were measured by Flow-FISH. The average length of telomere repeats over L4063 and L4064 during Gen 3 and Gen 3.1 were measured using Flow-FISH techniques. The measurement of the Relevant Telomere Length (RTL) was calculated using the telomere PNA kit/FITC from DAKO for flow cytometry analysis. Telomere analysis was performed. The telomere length in the sample was compared to that in the control cell line (1301 leukemia). The control cell line is a tetraploid cell line with long stable telomeres that allow calculation of relative telomere lengths. The Gen 3 and Gen 3.1 processes assessed in both tumors showed comparable telomere length.

TCR V beta lineage analysis

To determine the clonal diversity of the cellular products produced during each procedure, the clonal diversity analysis of the final TIL product was analyzed by sequencing the CDR3 portion of the T cell receptor.

Three parameters were compared between three conditions:

● Diversity index of unique CDR3 (uCDR 3)

● Sharing uCDR3%

● For uCDR first 80%:

comparison shared uCDR copies%

Frequency of the more distinct clonotypes

Control and Gen 3.1 test, TIL collects the percentage of shared unique CDR3 sequences on cell products: 975 sequences were shared between Gen 3 and Gen 3.1 test end products, equivalent to 88% of the first 80% from the Gen 3 unique CDR3 sequence shared with Gen 3.1.

Control and Gen 3.1 test, TIL collection cell products shared the percentage of unique CDR3 sequences: 2163 sequences were shared between Gen 3 and Gen 3.1 test end products, equivalent to 87% of the first 80% from the Gen 3 unique CDR3 sequence shared with Gen 3.1.

The number of unique CD3 sequences identified in 1 x 10 ⁶ cells from day 16 collection was used for different procedures. Based on the number of unique peptide CDRs in the sample, the Gen 3.1 test conditions showed slightly higher clonal diversity compared to Gen 3.0.

Xia Nongshang (Shannon entopy) diversity index is a reliable and commonly used comparative measure, since in both tumors, gen 3.1 conditions show slightly higher diversity compared to the Gen 3 process, indicating that the TCRV β lineage of Gen 3.1 test conditions is more polyclonal than the Gen 3.0 process.

Furthermore, the TCRV beta lineage of Gen3.1 test conditions showed more than 87% overlap with the corresponding lineage of Gen 3.0 process on tumors L4063 and L4064.

Gen3.1 test L4064 IL-2 concentration values on the reactivation day spent medium were lower than expected (similar to Gen3.1 control and Gen 3.0 conditions).

Low values may be due to pipetting errors, but since few samples are collected, it is not possible to repeat the analysis.

Conclusion (d). Gen 3.1 test conditions (including feeder cells and OKT-3) at day 0 showed higher TVC cell doses at day 16 of collection compared to Gen 3.0 and Gen 3.1 controls. The TVC of the final product under Gen 3.1 test conditions is about 2.5 times higher than Gen 3.0.

For both tumor samples tested, gen 3.1 test conditions with OKT-3 and feeder cells added on day 0 reached the maximum capacity of the flask at the time of collection. Under these conditions, if a maximum of 4 flasks were started on day 0, the final cell dose could be between 80-100×10 ⁹ TILs.

All quality attributes, such as phenotypic characterization, including purity of final TIL product, depletion, activation and memory markers, were maintained between Gen3.1 test and Gen 3.0 process.

In both tumors analyzed, IFN-. Gamma.production of the final TIL product in Gen 3.1 with feeder cells and OKT-3 added on day 0 was 3-fold higher than Gen 3.0, indicating that the Gen 3.1 process produced an effective TIL product.

No differences in glucose or lactate content were observed under each test condition. Under various medium conditions, no difference in glutamine and ammonia between Gen 3.0 and Gen 3.1 processes was observed. The lower glutamine content in the medium did not limit cell growth, indicating that the addition of GlutaMax ^TM alone to the medium was sufficient to provide the nutrients required for cell proliferation.

The scale up was longitudinal on day 11 and day 10, respectively, showed no significant difference in the number of cells reached on the day of collection of the process, and in both cases, the metabolite consumption during the whole process was comparable. This observation suggests that the Gen 3.0 optimization process may have flexibility in terms of treatment days, thereby facilitating flexibility in process scheduling.

The Gen 3.1 procedure with feeder cells and OKT-3 added on day 0 showed higher clonal diversity compared to Gen 3.0, as measured by CDR3 TCRab sequence analysis.

Fig. 32 depicts an example of a Gen 3 process (Gen 3 optimization process). Standard medium and CTS Optimizer serum-free medium can be used for TIL amplification in the Gen 3 optimization procedure. In the case of CTS Optimizer serum-free medium, it is recommended to increase the final concentration of GlutaMax ^TM on the medium to 4mM.

Example 11: preparation and characterization of modified TIL with Membrane-anchored IL-15 and IL-21 immunomodulatory fusion proteins

Materials and methods

Virus preparation and T cell transduction

To prepare lentiviruses, the pLenti-vector containing the tethered cytokine gene sequence (Table 58) and a packaging helper vector (VSV-G, gag/Pol) were co-transfected into 293T cells. Lentivirus supernatants were collected from day 2-3 culture supernatants and then ultracentrifuged (120,000 g) to concentrate lentivirus for TIL transduction. Pre-REP cells were stimulated with TransACT (1:100) for 2 days prior to T cell transduction. 1E5 activated pre-REP cells were added to a 48-well plate pre-coated with recombinant human fibrin fragments (Retronectin) along with concentrated lentivirus. Two days after gene transduction, pre-REP cells were collected for REP process or other phenotypic characterization and functional analysis.

Table 58: membrane anchored IL-15 and IL-21 fusion protein DNA sequences

Flow cytometry

To examine MIL-15 expression in transduced cells, transduced cells were stained using biotin-conjugate IL-15 (biolgend, san Diego, calif.) plus streptavidin-BV 421 (bioleged, san Diego, calif.). To examine the mIL-21 expression, transduced cells were stained with PE-conjugated IL-21 antibodies. To examine T cell division, T cells were pre-labeled with CELLTRACE ^TM violet cell proliferation dye (ThermoFisher scientific, waltham, MA). After 5 days, T cell division was analyzed in ex vivo cultures in the presence or absence of IL-2 (20 IU/mL). To examine T cell activation and differentiation, phenotypes were performed using antibodies from the bidi biosciences, biolegend, sameifeier. Data were acquired with a BioRad ZE5 flow cytometer and analyzed with FlowJo software (FlowJo, LLC, ashland, OR).

T cell count and viability

REP cells were collected after TIL expansion. Viable cell numbers were assessed by AOPI analysis using a Cellometer K2 cell counter (Nexcelom, lawrence, mass.).

Pre-REP and REP procedures

Human tumor samples were dissected into approximately 3mm pieces and incubated in Grex with recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were collected for 2 days of activation and 2 days of lentiviral transduction. The transduced pre-REP cells were then re-propagated with irradiated PBMCs, anti-CD 3 antibodies and 3,000iu/mL IL-2 during REP expansion for an additional 11 days.

Results

Expression of mIL-15/mIL-21 after Gene transduction

Following gene transduction, transduced pre-REP cells were expanded ex vivo with 300IU/mL IL-2 for additional 5 days. By flow cytometry staining, transduced pre-REP cells showed expression of mll-15 and mll-21. As shown in FIG. 38A, there was expression of 54.8% mIL-15 in mIL-15 lentiviral transduced TIL, 53.5% mIL-21 in mIL-21 transduced TIL, and 36.5% mIL-15/mIL-21 double positive cells in mIL-15/IL21 transduced TIL. As negative control, non-transduced cells were used.

MIL-15 pre-REP cells showed sustained activation of pSTAT5 signaling and increased proliferation

After validation of surface cytokine expression, T cell functionality was examined. Phosphorylation of STAT5 is a marker of T cell activation following gamma chain cytokine stimulation. Cells transduced with mIL-15, mIL-21 and mIL-15/IL-21 lentiviruses showed sustained pSTAT5 signaling under serum starvation conditions, indicating that mIL-15 is functional. As shown in FIG. 38B, mIL-15 is superior to mIL-21 in inducing pSTAT5 activation. In addition, the proliferation of cells of mIL-15 lentiviral transduced TILs was examined by CELLTRACE proliferation assay. TILs previously UV-labeled with CELLTRACE were incubated ex vivo for 5 days in the presence or absence of IL-2. As shown in FIG. 38C, mIL-15TIL showed better proliferative capacity than mIL-21 or mIL-15/IL-21TIL in the presence or absence of IL-2. mIL-15TIL can proliferate in the absence of IL-2, indicating that mIL-15 can provide a proliferation signal in place of IL-2.

Expression of mIL-15/mIL-21 after REP Process

After lentiviral transduction, TIL was amplified for 11 days in the REP process. The surface expression of membrane-bound cytokine receptors was then assessed by flow cytometry. As shown in FIG. 39A, there was expression of 31.1% mIL-15 in mIL-15TIL, 63% mIL-21 in mIL-21TIL, and 10.7% mIL-15/mIL-21 double positive cells in mIL-15/IL21 TIL. As negative control, non-transduced cells were used.

Expression of mIL-15/mIL-21 promotes CD8T cell expansion during REP

In the REP cells collected, an increase in the percentage of CD8+ T cells was detected in TILs transduced with mIL-15, mIL-21 and mIL-15/IL-21 lentiviruses (FIG. 39B). This is consistent with previous reports that demonstrate that IL-15 and IL-21 act as potent stimulators that preferentially stimulate proliferation and survival of CD 8T cells.

Phenotype of mIL-15/mIL-21 expressing REP TIL

To characterize the immunomodulatory effects of mIL-15 and mIL-21 on REP cells, phenotypes were performed with freshly thawed REP cells in gated CD8+CD3+ T cells (FIG. 40) and CD4+CD3+ T cell subsets (FIG. 41). We found that mbIL-15 significantly inhibited CD25 (IL-2Rα) expression. Like IL-2, IL-15 competes in using IL-2Rβ and IL-2Rγ, indicating a negative feedback mechanism for regulating CD 25. In addition, mIL-15 appears to activate T cells characterized by higher TIM3, TOX expression, whereas IL-21 shows antagonism. In addition, both mIL-15 and mIL-21 inhibit Eomes's expression. No significant differences in the expression of other surface markers (e.g., PD-1, CD27, CXCR3, etc.) were observed.

Example 12: preparation and characterization of modified TIL with Membrane-anchored IL-15 and IL-21 immunomodulatory fusion proteins

For this study, a modified TIL with membrane-anchored IL-15 and/or IL-21 immunomodulatory fusion proteins would be prepared, wherein membrane-anchored immunomodulatory IL-15 and IL-21 expression is controlled using the NFAT promoter comprising an NFAT response element linked to a minimal human IL-2 promoter, see, e.g., table 59. The modified TIL will be characterized as follows.

Materials and methods

Virus preparation and T cell transduction

To prepare lentiviruses, the pLenti-vector containing the tethered cytokine gene sequence (Table 59) and a packaging helper vector (VSV-G, gag/Pol) were co-transfected into 293T cells. Lentivirus supernatants were collected from day 2-3 culture supernatants and then ultracentrifuged (120,000 g) to concentrate lentivirus for TIL transduction. Pre-REP cells were stimulated with TransACT (1:100) for 2 days prior to T cell transduction. 1E5 activated pre-REP cells were added to a 48-well plate pre-coated with recombinant human fibrin fragments along with concentrated lentivirus. Two days after gene transduction, pre-REP cells were collected for REP process or other phenotypic characterization and functional analysis.

Table 59: membrane anchored IL-12, IL-15 and IL-21DNA sequences (with NFAT promoter)

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Flow cytometry

T cell count and viability

Pre-REP and REP procedures

Human tumor samples were dissected into approximately 3mm pieces and incubated in Grex with recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were collected for 2 days of activation and 2 days of lentiviral transduction. The transduced pre-REP cells were then further expanded with irradiated PBMCs, anti-CD 3 antibodies and 3,000iu/mL IL-2 during REP expansion for 11 days.

Results

Expression of mIL-15/mIL-21 after Gene transduction

Following gene transduction, transduced pre-REP cells were expanded ex vivo with 300IU/mL IL-2 for additional 5 days. MIL-15 and mIL-21 expression of transduced pre-REP cells was assessed by flow cytometry staining. As negative control, non-transduced cells were used.

After validation of surface cytokine expression, T cell functionality was examined. Specifically, phosphorylation of STAT5 of the modified TIL was assessed under serum starvation conditions to determine whether the modified TIL expressed functional mll-15 and/or IL-21. In addition, the proliferation of cells of mIL-15 lentiviral transduced TILs was examined by CELLTRACE proliferation assay.

Expression of mIL-15/mIL-21 after REP Process

After lentiviral transduction, TIL was amplified for 11 days in the REP process and then surface expression of membrane-bound cytokine receptors was assessed by flow cytometry. The collected REP cells were evaluated against cd8+ T cells. Previous reports demonstrated that IL-15 and IL-21 act as potent stimulators that preferentially stimulate proliferation and survival of CD 8T cells.

Phenotype of mIL-15/mIL-21 expressing REP TIL

To characterize the immunomodulatory effects of mIL-15 and mIL-21 on REP cells, phenotypes were performed with freshly thawed REP cells in gated CD8+CD3+ T cells and CD4+CD3+ T cell subsets. The following expression will be evaluated: CD27, PD1, TIM3, CD62L, TOX, T-bet, CD38, CXCR3, eomes, and CD25.

Example 13: preparation and characterization of modified TIL with Membrane-anchored IL-2 and IL-12 immunomodulatory fusion proteins

Four cryopreserved TIL populations were thawed and cell numbers and viability were assessed using a Cellaca MX counter. The TIL is then resuspended and mRNA encoding membrane anchored IL-2 and/or IL-12 (mbiL-2 and mbiL 12) is delivered into a subset of the TIL using the SQZ method (i.e., "squeeze (squeeze)"). Exemplary mbIL-2 and mbIL-12 constructs are depicted in figure 37. The following TIL subpopulations will be evaluated: 1) No SQZ; 2) mRNA-free; 3) mbIL-2mRNA; 4) mbIL-12mRNA; and 5) mbIL-2+mbil-12mRNA. After extrusion, viability and number of cells were again assessed using Cellaca MX counter. The cells were then resuspended and cultured in CM2 medium for 4 days in the presence or absence of 300IU/mL IL-2. Cells were collected daily to determine cell number, viability and surface marker expression, including (but not limited to): mbIL-2 and mbIL-12. Following extrusion, cells will also be stimulated with TransAct in the presence or absence of 300IU/mL IL-2. Cell numbers, viability and marker expression will also be assessed as will IFNg and TNFa levels. Some TILs will also be cryopreserved after extrusion and thawed after 1 week to analyze the expression of mbIL-2 and mbIL-12 after 1,2, 3, and 4 days of culture in the presence or absence of IL-2. This will allow an assessment of whether the extruded and cryopreserved cells retain the same amount of membrane bound cytokine expression as compared to fresh cells.

The functional effect of cells extruded with mbIL-2 and mbIL-12 was evaluated in an in vitro cytotoxicity assay using KILR THP-1 cells. The extruded TIL was co-cultured with KILR THP-1 cells at a ratio of 10:1 in the presence or absence of 300IU/mL IL-2. After 24 hours, cytotoxicity was assessed by adding substrate to the culture plate. In a separate experimental set, the extruded and control TILs were co-cultured with KILR THP-1 cells for 3 days in the presence or absence of 300IU/mL IL-2. After this incubation period, TIL numbers, survival and phenotypes were assessed.

After this initial set of in vitro experiments, PDX models were run in NSG and hIL-2NOG mice with control and TIL extruded with mbiL-2 and mbiL-12. For these experiments, cryopreserved melanoma TIL and paired PDX tumor cells will be used. Briefly, PDX tumor cells were inoculated in NSG and hIL-2NOG mice. After tumor establishment, tumor bearing mice were vaccinated with control group and extruded TIL (corresponding to PDX model) at 10E6 cells/mouse by tail vein injection. Tumor and mouse body weights were measured once every two weeks.

After the initial proof of concept (POC) experiment was completed as described above, two engineering operations were performed using the Gen2 procedure described in example 7 above, followed by a cell extrusion process (as described above) to transfect the resulting TIL population with mbIL-2mRNA and/or mbIL-12 mRNA. TIL was assessed for cell count and viability before and after extrusion, and marker expression was assessed after extrusion with or without IL-2 and for 4 days.

***

The above examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes of carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the level of skill of those skilled in the art.

All headings and chapter designations are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from the different titles and chapters as desired in accordance with the spirit and scope of the present invention as described herein.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

As will be apparent to those skilled in the art, various modifications and variations can be made thereto without departing from the spirit and scope of the application. The particular embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of tumor-infiltrating lymphocytes (TILs), wherein, optionally, the patient or subject has received at least one prior therapy,

A portion of the TILs are modified TILs such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

2. A method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of modified tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Obtaining and/or receiving a first population of TILs from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) Adding a first TIL population to a closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2, producing a second population of TILs, wherein the first amplification is performed in a closed container providing a first gas permeable surface area, the first amplification being performed for about 3 to 14 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(d) Performing a second amplification by supplementing a cell culture medium of a second population of TILs with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a third population of TILs, wherein the second amplification is performed for about 7 to 14 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(h) Administering to the subject a therapeutically effective dose of a third population of TILs from the infusion bag in step (g); and (i) modifying a portion of the TILs at any time prior to administration of (h) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface film.

3. A method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(b) Adding tumor fragments into the closed system;

(c) Performing a first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2, producing a second population of TILs, wherein the first amplification is performed in a closed container providing a first gas permeable surface area, the first amplification being performed for about 3 to 11 days to obtain the second population of TILs, the transition from step (b) to step (c) being performed without opening the system;

(d) Performing a second amplification by supplementing a cell culture medium of a second population of TILs with additional IL-2, OKT-3 and Antigen Presenting Cells (APC), resulting in a third population of TILs, wherein the second amplification is performed for about 7 to 11 days to obtain the third population of TILs, the second amplification being performed in a closed container providing a second gas permeable surface area, the transition from step (c) to step (d) being performed without opening the system;

(h) Administering to the subject a therapeutically effective dose of a third population of TILs from the infusion bag in step (g); and

4. A method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(B) Adding a first TIL population to a closed system;

5. A method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of modified tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(h) Administering a therapeutically effective dose of a third TIL population from the infusion bag in step (g) to a subject or patient having cancer; and

6. A method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(c) Contacting a first population of TILs with a first cell culture medium;

(f) Collecting a third TIL population;

(H) Modifying a portion of the TILs at any time prior to administration (g) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

7. A method of treating cancer in a patient or subject in need thereof, the method comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of:

(a) Resecting a tumor from a cancer of a subject or patient, the tumor comprising a first population of TILs, optionally by surgical resection, needle aspiration biopsy, coarse needle biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from the cancer;

(b) Breaking up the tumor into tumor fragments;

(c) Contacting tumor fragments with a first cell culture medium;

(f) Collecting a third TIL population;

8. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising:

(c) Initiating a first expansion by culturing a PD-l enriched population of TILs in a first cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed in a vessel comprising a first gas permeable surface region, wherein initiating the first expansion is performed for a first period of time of about 1 to 7 days or about 1 to 8 days to obtain the second population of TILs, the second population of TILs being greater in number than the first population of TILs;

(d) Performing a rapid second amplification by culturing a second population of TILs in a second medium comprising IL-2, OKT-3 and APCs, producing a population of therapeutic TILs, wherein the number of APCs added in the rapid second amplification is at least twice the number of APCs added in step (b), the rapid second amplification being performed for a second period of about 1 to 11 days to obtain a population of therapeutic TILs, the third population of TILs being the population of therapeutic TILs, wherein the rapid second amplification is performed in a container having a second gas permeable surface area;

(e) Collecting the therapeutic TIL population obtained from step (d);

9. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(a) Obtaining and/or receiving a first TIL population of tumors from a cancer resection from a subject or patient by processing a tumor sample obtained from the tumor into a plurality of tumor fragments;

(b) Adding a first TIL population to a closed system;

10. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(a) Obtaining a first TIL population from a tumor resected from a cancer of a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments;

(b) Adding tumor fragments into the closed system;

(f) Transferring the collected third TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) is performed without opening the system; and

11. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(B) Adding a first TIL population to a closed system;

12. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(b) Adding tumor fragments into the closed system;

13. The method of any one of claims 9 to 12, wherein the first amplification is divided into a first step and a second step, the method further comprising a first step of performing the first amplification by culturing the first population of TILs in a cell culture medium comprising IL-2, producing TILs from the tumor fragments or samples, separating the TILs remaining in the tumor fragments or samples from the TILs from the tumor fragments or samples, optionally digesting the tumor fragments or samples to produce tumor digests; and a second step of performing the first amplification by culturing the remaining TILs in the tumor fragments or samples or tumor digests in a cell culture medium, resulting in a second population of TILs.

14. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(b) Contacting a first population of TILs with a first cell culture medium;

(d) Performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD 3 antibody) and APC; the rapid amplification is performed for a period of time less than 14 days, alternatively, the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the start of the rapid second amplification;

(e) Collecting a third TIL population; and

15. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of:

(b) Fragmenting the tumor into tumor fragments;

(c) Contacting tumor fragments with a first cell culture medium;

(e) Performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD 3 antibody) and APC; the rapid amplification is performed for a period of time less than 14 days, alternatively, the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the start of the rapid second amplification;

(f) Collecting a third TIL population; and

16. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising:

(1) Obtaining and/or receiving a first TIL population from a tumor resected from a cancer of a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments;

(2) Initiating a first expansion by culturing a first population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, and optionally comprising Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed for a first period of about 1 to 7 days or about 1 to 8 days to obtain the second population of TILs, the number of the second population of TILs being greater than the first population of TILs;

(3) Performing a rapid second amplification by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and APC, producing a third population of TILs, wherein the rapid second amplification is performed for a second period of time of about 1 to 11 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs;

(4) Collecting the therapeutic TIL population obtained from step (c); and

(5) Modifying a portion of the TILs at any time before or after collection in step (d) such that each of the modified TILs comprises an immunomodulatory composition associated with its surface membrane.

17. The method of claim 16, wherein in step (b), the cell culture medium further comprises Antigen Presenting Cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

18. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising:

(a) Initiating a first expansion by culturing a first population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, and optionally Antigen Presenting Cells (APCs), resulting in a second population of TILs, the first population of TILs obtainable by processing a tumor sample of a tumor resected from a cancer of a subject into a plurality of tumor fragments; wherein initiating the first amplification is performed in a vessel having a first gas permeable surface region, the initiating the first amplification being performed for a first period of time of about 1 to 7 days or about 1 to 8 days to obtain a second population of TILs, the second population of TILs being greater in number than the first population of TILs;

(c) Collecting the therapeutic TIL population obtained from step (b); and

19. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising:

(a) Initiating a first expansion by culturing a first population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, and optionally Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed for a first period of about 1 to 7 days or about 1 to 8 days to obtain the second population of TILs, the number of the second population of TILs being greater than the first population of TILs;

(b) Performing a rapid second amplification by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and APC, producing a third population of TILs, wherein the rapid second amplification is performed for a second period of time of about 1 to 11 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs;

(c) Collecting the therapeutic TIL population obtained from step (b); and

20. The method of claim 19, wherein in step (a), the cell culture medium further comprises Antigen Presenting Cells (APCs), and the number of APCs in the medium in step (c) is greater than the number of APCs in the medium in step (b).

21. The method of any one of claims 14 to 18, wherein initiating the first expansion is divided into a first step and a second step, the method further comprising performing the first step of initiating the first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2, producing TILs from tumor fragments or samples, separating the TILs remaining in the tumor fragments or samples from TILs from tumor fragments or samples, optionally digesting the tumor fragments or samples to produce tumor digests; and performing a second step of initiating the first expansion by culturing the remaining TILs in the tumor fragments or samples or tumor digests in cell culture medium, resulting in a second population of TILs.

22. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising:

(a) Obtaining and/or receiving a first TIL population from a tumor sample obtained from one or more small biopsy, coarse needle biopsy, or needle biopsy of a tumor from a cancer of a subject by culturing the tumor sample in a first cell culture medium comprising IL-2 for about 3 days;

(b) Initiating a first expansion by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed in a vessel having a first gas permeable surface region, the initiating the first expansion being performed for a first period of about 7 or 8 days to obtain the second population of TILs, the second population of TILs being greater in number than the first population of TILs;

(c) Generating a third TIL population by performing a rapid second amplification by supplementing a second cell culture medium of a second TIL population with additional IL-2, OKT-3, and APCs, wherein the number of APCs added in the rapid second amplification is at least twice the number of APCs added in step (b), the rapid second amplification being performed for a second period of about 11 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, the rapid second amplification being performed in a vessel having a second gas permeable surface area;

(d) Collecting the therapeutic TIL population obtained from step (c);

23. A method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising:

(b) Initiating a first expansion by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a second population of TILs, wherein initiating the first expansion is performed for a first period of time of about 7 or 8 days to obtain the second population of TILs, the number of the second population of TILs being greater than the first population of TILs;

(c) Performing a rapid second amplification by contacting the second population of TILs with a third cell culture medium comprising IL-2, OKT-3, and APC, producing a third population of TILs, wherein the rapid second amplification is performed for a second period of about 11 days to obtain the third population of TILs, the third population of TILs being a therapeutic population of TILs;

(d) Collecting the therapeutic TIL population obtained from step (c); and

24. The method of any one of claims 1 to 18 and 21 to 23, wherein the cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

25. A method of expanding T cells, the method comprising:

(a) Performing an initial first expansion of the first T cell population obtained from the donor by culturing the first T cell population to effect growth and initiate activation of the first T cell population;

(b) After the activation of the first T cell population initiated in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population to effect growth and enhance activation of the first T cell population to obtain a second T cell population;

(c) Collecting a second T cell population; and

26. A method of expanding T cells, the method comprising:

(a) Performing an initial first expansion of a first T cell population from a tumor sample obtained from more than one small biopsy, coarse needle biopsy, or needle biopsy of a tumor in a donor by culturing the first T cell population to achieve growth and initiating activation of the first T cell population;

(c) Collecting a second T cell population; and

27. A method of amplifying Peripheral Blood Lymphocytes (PBLs) from peripheral blood, the method comprising the steps of:

(b) PBMCs were cultured in a culture comprising a first cell culture medium for a period of time selected from the group consisting of: about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, and about 14 days to effect expansion of Peripheral Blood Lymphocytes (PBLs) from the PBMCs, the first cell culture medium having a combination of IL-2, an anti-CD 3/anti-CD 28 antibody, and a first antibiotic;

(c) Collecting PBL from the culture in step (b); and

28. The method of claim 27, wherein the patient has been pre-treated with ibrutinib or another interleukin-2 inducible T cell kinase (ITK) inhibitor.

29. The method of claim 26 or 28, wherein the patient is refractory to treatment with ibrutinib or another ITK inhibitor.

30. The method of any one of claims 2 to 29, wherein the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor moiety.

31. The method of claim 30, wherein the one or more immunomodulators comprise one or more cytokines.

32. The method of claim 31, wherein the one or more cytokines comprises IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, ifnγ, TNFa, ifnα, ifnβ, GM-CSF, or GCSF, or a variant thereof.

33. The method of claim 32, wherein the one or more cytokines comprise IL-12.

34. The method of claim 33, wherein the IL-12 comprises a human IL-12p35 subunit linked to a human IL-12p40 subunit.

35. The method of claim 34, wherein the human IL-12p35 subunit has the amino acid sequence of SEQ ID No. 247 and the human IL-12p40 subunit has the amino acid sequence of SEQ ID No. 248.

36. The method of claim 32, wherein the one or more cytokines comprise IL-15.

37. The method of claim 36, wherein the IL-15 is human IL-15.

38. The method of claim 37, wherein the human IL-15 has the amino acid sequence of SEQ ID No. 258.

39. The method of claim 32, wherein the one or more cytokines comprise IL-18.

40. The method of claim 39, wherein the IL-18 is human IL-18.

41. The method of claim 40, wherein the human IL-18 has the amino acid sequence of SEQ ID NO:269 or SEQ ID NO: 270.

42. The method of claim 32, wherein the one or more cytokines comprise IL-21.

43. The method of claim 42, wherein the IL-21 is human IL-21.

44. The method of claim 43, wherein the human IL-21 has the amino acid sequence of SEQ ID NO: 271.

45. The method of claim 30, wherein the one or more immunomodulators comprise a CD40 agonist.

46. The method of claim 45, wherein the CD40 agonist is an anti-CD 40 binding domain or CD40L.

47. The method of claim 46, wherein the CD40 agonist is a CD40 binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL).

48. The method of claim 47, wherein the VH and VL of the CD40 binding domain are selected from the group consisting of:

a. VH having the amino acid sequence of SEQ ID No. 274 and VL having the amino acid sequence of SEQ ID No. 275;

b. VH having the amino acid sequence of SEQ ID No. 277 and VL having the amino acid sequence of SEQ ID No. 278;

c. VH having the amino acid sequence of SEQ ID No. 280 and VL having the amino acid sequence of SEQ ID No. 281; and

D. VH having the amino acid sequence of SEQ ID No. 283 and VL having the amino acid sequence of SEQ ID No. 284.

49. The method of claim 47 or 48, wherein the CD40 binding domain is an scFv.

50. The method of claim 46, wherein the CD40 agonist is human CD40L having the amino acid sequence of SEQ ID NO. 273.

51. The method of any one of claims 30 to 50, wherein the membrane-anchored immunomodulatory fusion protein is according to the formula from N-terminus to C-terminus: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety.

52. The method of any one of claims 30 to 51, wherein the cell membrane anchor portion comprises a CD8a transmembrane-intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain.

53. The method of claim 52, wherein the cell membrane anchor portion comprises a B7-1 transmembrane domain.

54. The method of claim 53, wherein the cell membrane anchor portion has the amino acid sequence of SEQ ID NO. 239.

55. The method of any one of claims 30 to 54, wherein the immunomodulatory composition comprises two or more different membrane-anchored immunomodulatory fusion proteins, each of the different membrane-anchored immunomodulatory fusion proteins each comprising a different immunomodulatory agent.

56. The method of claim 55, wherein the different immunomodulator is selected from the group consisting of: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF, GCSF, or variants thereof, and CD40 agonists.

57. The method of claim 56, wherein said different immunomodulator is selected from the group consisting of: IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12.

58. The method of any one of claims 30 to 57, wherein the modification comprises introducing a heterologous nucleic acid encoding a fusion protein into a portion of the TIL, the fusion protein being expressed on the surface of the modified TIL.

59. The method of claim 58, wherein the heterologous nucleic acid is introduced into the genome of the modified TIL using one or more methods selected from the group consisting of: CRISPR method, TALE method, zinc finger method and combinations thereof.

60. The method of any one of claims 2 to 29, wherein the immunomodulatory composition comprises a fusion protein comprising one or more immunomodulatory agents linked to a TIL surface antigen binding domain.

61. The method of claim 60, wherein the one or more immunomodulators comprise one or more cytokines.

62. The method of claim 61, wherein the one or more cytokines comprises IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNγ, TNFa, IFNα, IFNβ, GM-CSF, or GCSF, or a variant thereof.

63. The method of claim 62, wherein the one or more cytokines comprise IL-12.

64. The method of claim 62, wherein the one or more cytokines comprise IL-15.

65. The method of claim 62, wherein the one or more cytokines comprise IL-21.

66. The method of any one of claims 60 to 65, wherein the TIL surface antigen binding domain comprises an antibody heavy chain variable domain and a light chain variable domain.

67. The method of any one of claims 60 to 66, wherein the TIL surface antigen binding domain comprises an antibody or fragment thereof.

68. The method of any one of claims 43 to 50, wherein the TIL surface antigen binding domain has an affinity ：CD45、CD4、CD8、CD3、CDlla、CDllb、CDllc、CD18、CD25、CD127、CD19、CD20、CD22、HLA-DR、CD197、CD38、CD27、CD196、CXCR3、CXCR4、CXCR5、CD84、CD229、CCR1、CCR5、CCR4、CCR6、CCR8、CCR10、CD 16、CD56、CD 137、OX40 or GITR for one or more of the following TIL surface antigens.

69. The method of any one of claims 60 to 68, wherein the modifying comprises incubating the fusion protein with a portion of the TIL under conditions that allow the fusion protein to bind to a portion of the TIL.

70. The method of any one of claims 2 to 29, wherein the immunomodulatory composition comprises a nanoparticle comprising a plurality of immunomodulatory agents.

71. The method of claim 70, wherein the plurality of immunomodulatory agents are covalently linked together by a degradable linker.

72. The method of claim 71, wherein the nanoparticle comprises at least one polymer, cationic polymer, or cationic block copolymer on a surface of the nanoparticle.

73. The method of any one of claims 70-72, wherein the one or more cytokines comprise IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, ifnγ, TNFa, ifnα, ifnβ, GM-CSF or GCSF or a variant thereof.

74. The method of claim 73, wherein the one or more cytokines comprise IL-12.

75. The method of claim 73, wherein the one or more cytokines comprise IL-15.

76. The method of claim 73, wherein the one or more cytokines comprise IL-21.

77. The method of any one of claims 70 to 76, wherein the nanoparticle is a liposome, a protein nanogel, a nucleotide nanogel, a polymer nanoparticle, or a solid nanoparticle.

78. The method of claim 77, wherein said nanoparticle is a nanogel.

79. The method of any one of claims 70 to 78, wherein the nanoparticle further comprises an antigen binding domain that binds more than one of the following antigens: CD45, CDlla (integrin α -L), CD 18 (integrin β -2), CD1lb, CD1lc, CD25, CD8 or CD4.

80. The method of any one of claims 70 to 79, wherein the modifying comprises attaching the immunomodulatory composition to a surface of a portion of the TIL.

81. The method of any one of claims 2 to 5 or 9 to 13, wherein TIL from the first amplification or TIL from the second amplification or both are modified.

82. The method of any one of claims 6 to 8 or 14 to 23, wherein TIL from the initial first amplification or TIL from the rapid second amplification or both are modified.

83. The method of any one of claims 2 to 5 or 9 to 13, wherein the modification is performed after the first amplification and before the second amplification.

84. The method of any one of claims 6 to 8 or 14 to 23, wherein the modification is performed after initiation of the first amplification and before rapid second amplification, or at both time points.

85. The method of any one of claims 2 to 5 or 9 to 13, wherein the modification is performed after the second amplification.

86. The method of any one of claims 6 to 8 or 14 to 23, wherein the modification is performed after the rapid second amplification.

87. The method of any one of claims 2 to 63, wherein the modification is performed after collection.

88. The method of any one of claims 2 to 5 or 9 to 13, wherein the first amplification is performed for a period of about 11 days.

89. The method of any one of claims 6-8 or 14-23, wherein the initiating first amplification is performed for a period of about 11 days.

90. The method of any one of claims 2 to 5 or 9 to 13, wherein in the first amplification the IL-2 is present in the cell culture medium at an initial concentration of between 1000IU/mL and 6000 IU/mL.

91. The method of any one of claims 6 to 8 or 14 to 23, wherein in initiating the first amplification, the IL-2 is present in the cell culture medium at an initial concentration of between 1000IU/mL and 6000 IU/mL.

92. The method of any one of claims 2 to 5 or 9 to 13, wherein in the second amplification step the IL-2 is present at an initial concentration of between 1000IU/mL and 6000IU/mL, and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

93. The method of any one of claims 6 to 8 or 14 to 23, wherein in the rapid second amplification step the IL-2 is present at an initial concentration of between 1000IU/mL and 6000IU/mL, and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

94. The method of any one of claims 2 to 5 or 9 to 13, wherein the first amplification is performed using a gas-permeable container.

95. The method of any one of claims 6 to 8 or 14 to 23, wherein the initiating first amplification is performed using a gas-permeable container.

96. The method of any one of claims 2 to 5 or 9 to 13, wherein the second amplification is performed using a gas-permeable container.

97. The method of any one of claims 6 to 8 or 14 to 23, wherein the rapid second amplification is performed using a gas-permeable container.

98. The method of any one of claims 2 to 5 or 9 to 13, wherein the first expanded cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof.

99. The method of any one of claims 6 to 8 or 14 to 23, wherein the cell culture medium that initiates the first expansion further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof.

100. The method of any one of claims 2 to 5 or 9 to 13, wherein the second expanded cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof.

101. The method of any one of claims 6 to 8 or 14 to 23, wherein the fast second expanded cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL-21 and combinations thereof.

102. The method of any one of claims 1 to 8, further comprising the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering TIL to the patient.

103. The method of claim 102, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for three days.

104. The method of claim 102, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day and fludarabine was administered at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for three days.

105. The method of claim 102, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: cyclophosphamide was administered at a dose of 60mg/m ²/day and fludarabine was administered at a dose of 25mg/m ²/day for two days, followed by fludarabine at a dose of 25mg/m ²/day for one day.

106. The method of any one of claims 103-105, wherein cyclophosphamide is administered with mesna (mesna).

107. The method of any one of claims 1-7 or 102-106, further comprising the step of beginning treatment of the patient with an IL-2 regimen the next day after administration of TIL to the patient.

108. The method of any one of claims 1-7 or 102-106, further comprising the step of beginning treatment of the patient with an IL-2 regimen on the same day as the administration of TIL to the patient.

109. The method of claim 107 or 108, wherein the IL-2 regimen is a high dose IL-2 regimen comprising 600,000 or 720,000IU/kg of aldesleukin or a biological analog or variant thereof, which regimen is administered as an intravenous infusion of 15 minute bolus injections every eight hours until tolerated.

110. The method of any one of claims 1 to 7 or 102 to 109, wherein a therapeutically effective population of TILs comprising about 2.3 x 10 ¹⁰ to about 13.7 x 10 ¹⁰ TILs is administered.

111. The method of any one of claims 6 to 8 or 14 to 23, wherein the initiating first amplification and the rapidly second amplification are performed for a period of time less than 21 days.

112. The method of any one of claims 6 to 8 or 14 to 23, wherein the initiating first amplification and the rapidly second amplification are performed for a period of 16 days or less.

113. The method of any one of claims 6 to 8 or 14 to 23, wherein the initial first amplification is performed for a period of time of 7 or 8 days or less.

114. The method of any one of claims 6 to 8 or 14 to 23, wherein the rapid second amplification is performed for a period of time less than 11 days.

115. The method of any one of claims 2 to 5 or 9 to 13, wherein the first amplification in step (c) and the second amplification in step (d) are each separately performed over a period of 11 days.

116. The method of any one of claims 2 to 5 or 9 to 13, wherein steps (a) to (f) are performed within about 10 days to about 22 days.

117. The method of any one of claims 2 to 116, wherein the modified TIL further comprises a genetic modification that causes silencing or reduction of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

118. The method of claim 117, wherein the one or more immune checkpoint genes is selected from ：PD-1、CTLA-4、LAG-3、HAVCR2(TIM-3)、Cish、TGFβ、PKA、CBL-B、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、BTLA、CD160、TIGIT、TET2、CD96、CRTAM、LAIR1、SIGLEC7、SIGLEC9、CD244、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT1、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2、GUCY1B3、TOX、SOCS1、ANKRD11 and BCOR.

119. The method of claim 117, wherein the one or more immune checkpoint genes are selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), cish, TGF beta and PKA.

120. The method of any one of claims 2 to 119, wherein the modified TIL further comprises a genetic modification that results in an enhancement of expression of one or more immune checkpoint genes in at least a portion of the population of therapeutic TILs, the one or more immune checkpoint genes selected from the group consisting of: CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1.

121. The method of any one of claims 117 to 120, wherein the genetic modification is performed using a programmable nuclease that mediates the generation of double-or single-stranded breaks at the one or more immune checkpoint genes.

122. The method of any one of claims 117 to 120, wherein the genetic modification is performed using one or more methods selected from the group consisting of: CRISPR method, TALE method, zinc finger method and combinations thereof.

123. The method of claim 122, wherein the genetic modification is performed using a CRISPR method.

124. The method of claim 123, wherein the CRISPR method is a CRISPR/Cas9 method.

125. The method of claim 122, wherein the genetic modification is performed using a TALE method.

126. The method of claim 122, wherein the genetic modification is performed using a zinc finger method.

127. The method of any one of claims 1to 23 or 81 to 116, wherein the modified TIL is modified to transiently express the immunomodulatory composition on the surface of a cell.

128. The method of claim 127, wherein the immunomodulatory composition comprises one or more membrane anchored immunomodulatory fusion proteins, each fusion protein comprising one or more immunomodulatory agents and a cell membrane anchor moiety.

129. The method of claim 128, wherein the one or more immunomodulators comprise one or more cytokines.

130. The method of claim 129, wherein the one or more cytokines comprise IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, ifnγ, TNFa, ifnα, ifnβ, GM-CSF or GCSF or a variant thereof.

131. The method of claim 130, wherein the one or more cytokines comprise IL-2.

132. The method of claim 131, wherein the IL-2 is human IL-2.

133. The method of claim 132, wherein the human IL-2 has the amino acid sequence of SEQ ID NO: 272.

134. The method of claim 130, wherein the one or more cytokines comprise IL-12.

135. The method of claim 134, wherein the IL-12 comprises a human IL-12p35 subunit linked to a human IL-12p40 subunit.

136. The method of claim 135, wherein the human IL-12p35 subunit has the amino acid sequence of SEQ ID No. 267 and the human IL-12p40 subunit has the amino acid sequence of SEQ ID No. 268.

137. The method of claim 130, wherein the one or more cytokines comprise IL-15.

138. The method of claim 137, wherein the IL-15 is human IL-15.

139. The method of claim 138, wherein the human IL-15 has the amino acid sequence of SEQ ID No. 258.

140. The method of claim 130, wherein the one or more cytokines comprise IL-18.

141. The method of claim 140, wherein the IL-18 is human IL-18.

142. The method of claim 141, wherein the human IL-18 has the amino acid sequence of SEQ ID No. 269 or SEQ ID No. 270.

143. The method of claim 130, wherein the one or more cytokines comprise IL-21.

144. The method of claim 143, wherein the IL-21 is human IL-21.

145. The method of claim 144, wherein the human IL-21 has the amino acid sequence of SEQ ID NO: 271.

146. The method of claim 128, wherein the one or more immunomodulators comprise a CD40 agonist.

147. The method of claim 146, wherein the CD40 agonist is an anti-CD 40 binding domain or CD40L.

148. The method of claim 147, wherein the CD40 agonist is a CD40 binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL).

149. The method of claim 148, wherein the VH and VL of the CD40 binding domain are selected from the group consisting of:

150. The method of claim 148 or 149, wherein the CD40 binding domain is an scFv.

151. The method of claim 46, wherein the CD40 agonist is human CD40L having the amino acid sequence of SEQ ID NO. 273.

152. The method of any one of claims 128-151, wherein the one or more membrane-anchored immunomodulatory fusion proteins are independently according to the formula from N-terminus to C-terminus: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety.

153. The method of any one of claims 128 to 152, wherein the cell membrane anchor portion comprises a CD8a transmembrane-intracellular domain, B7-1 transmembrane domain, B7-2 transmembrane domain, or CD8a transmembrane domain.

154. The method of claim 153, wherein the cell membrane anchor portion comprises a B7-1 transmembrane domain.

155. The method of claim 154, wherein the cell membrane anchor portion has the amino acid sequence of SEQ ID No. 239.

156. The method of any one of claims 128-155, wherein the immunomodulatory composition comprises two or more different membrane-anchored immunomodulatory fusion proteins, each of the different membrane-anchored immunomodulatory fusion proteins each comprising a different immunomodulatory agent.

157. The method of claim 156, wherein the different immunomodulatory agent is selected from the group consisting of: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF, GCSF, or variants thereof, and CD40 agonists.

158. The method of claim 157, wherein the different immunomodulatory agent is selected from the group consisting of: IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12.

159. The method of any one of claims 128-158, wherein the modification comprises introducing a heterologous nucleic acid encoding a fusion protein into a portion of the TIL, the fusion protein being expressed on the surface of the modified TIL.

160. The method of claim 159, wherein the heterologous nucleic acid is introduced into the genome of the modified TIL using one or more methods selected from the group consisting of: CRISPR method, TALE method, zinc finger method and combinations thereof.

161. The method of any of claims 128-158, wherein the modified TIL is modified by transfecting the TIL with a nucleic acid encoding the fusion protein.

162. The method of claim 161, wherein the nucleic acid is RNA.

163. The method of claim 162, wherein the RNA is mRNA.

164. The method of claim 163, wherein TIL is transfected with the mRNA by electroporation.

165. The method of claim 164, wherein after the first amplification and before the second amplification, the TIL is transfected with the mRNA by electroporation.

166. The method of claim 164, wherein, prior to the first amplification, TIL is transfected with the mRNA by electroporation.

167. The method of claim 161, wherein the modified TIL is transfected with a nucleic acid encoding the fusion protein by temporarily disrupting a cell membrane of the TIL using a microfluidic device, thereby allowing transfection of the nucleic acid.

168. The method of any one of claims 163-167, wherein the method further comprises activating TIL by incubation with an anti-CD 3 agonist prior to transfection of TIL with the mRNA.

169. The method of claim 168, wherein the anti-CD 3 agonist is OKT-3.

170. The method of claim 168 or 169, wherein TIL is activated by incubating TIL with the anti-CD 3 agonist for about 1 to 3 days prior to transfection of TIL with the mRNA.

171. A composition comprising the modified TIL of any of claims 1-131.

172. A pharmaceutical composition comprising the modified TIL of any one of claims 1-131 and a pharmaceutically acceptable carrier.

173. The method of claim 30, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-2.

174. The method of claim 30, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-15.

175. The method of claim 30, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-18.

176. The method of claim 30, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-21.

177. The method of claim 30, wherein the modified TIL comprises a first membrane-anchored immunomodulatory fusion protein and a second membrane-anchored immunomodulatory fusion protein.

178. The method of claim 177, wherein the first membrane-anchored immunomodulatory fusion protein comprises IL-15 and the second membrane-anchored immunomodulatory fusion protein comprises IL-21.

179. The method of claim 177 or 178, wherein expression of the first membrane-anchored immunomodulatory fusion protein and the second immunomodulatory fusion protein is controlled by an NFAT promoter in the modified TIL.

180. The method of claim 30, wherein the one or more membrane-anchored immunomodulatory fusion proteins are independently according to the formula from N-terminus to C-terminus: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety.

181. The method of claim 180, wherein IA is a cytokine.

182. The method of claim 180, wherein IA is selected from the group consisting of: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof.

183. The method of claim 180, wherein IA is IL-2.

184. The method of claim 180, wherein IA is IL-12.

185. The method of claim 180, wherein IA is IL-15.

186. The method of claim 180, wherein IA is IL-21.

187. The method of claim 30, wherein the one or more membrane-anchored immunomodulatory fusion proteins are independently according to the formula from N-terminus to C-terminus: S1-IA1-L1-C1-L2-S2-IA2-L3-C2, wherein S1 and S2 are each independently a signal peptide, IA1 and IA2 are each independently an immunomodulator, L1 to L3 are each independently a linker, and C1 and C2 are each independently a cell membrane anchor moiety.

188. The method of claim 187, wherein S1 is the same as S2.

189. The method of claim 187 or 188, wherein C1 is the same as C2.

190. The method of any one of claims 187-189, wherein L2 is a cleavable linker.

191. A method in accordance with claim 190, wherein L2 is a furin cleavable linker.

192. The method of any one of claims 187-191, wherein IA1 and IA2 are each independently a cytokine.

193. The method of any one of claims 187-191, wherein IA1 and IA2 are each independently selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof.

194. The method of any one of claims 187-191, wherein IA1 and IA2 are each independently selected from IL-2 and IL-12, provided that one of IA1 and IA2 is IL-2 and the other is IL-12.

195. The method of any one of claims 187-191, wherein IA1 and IA2 are each independently selected from IL-15 and IL-21, provided that one of IA1 and IA2 is IL-15 and the other is IL-21.

196. The method of any one of claims 1-126 or 173-195, wherein the modified TIL is genetically modified to express the immunomodulatory composition on a surface of a cell.

197. The method of claim 196, wherein the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor moiety.

198. The method of claim 197, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-2.

199. The method of claim 197, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-15.

200. The method of claim 197, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-18.

201. The method of claim 197, wherein the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-21.

202. The method of claim 197, wherein the modified TIL comprises a first membrane-anchored immunomodulatory fusion protein and a second membrane-anchored immunomodulatory fusion protein.

203. The method of claim 202, wherein the first membrane-anchored immunomodulatory fusion protein comprises IL-15 and the second membrane-anchored immunomodulatory fusion protein comprises IL-21.

204. The method of claim 202 or 203, wherein expression of the first membrane-anchored immunomodulatory fusion protein and the second immunomodulatory fusion protein is controlled by an NFAT promoter in a modified TIL.

205. The method of claim 197, wherein the one or more membrane-anchored immunomodulatory fusion proteins are independently according to the formula from N-terminus to C-terminus: S-IA-L-C, wherein S is a signal peptide, IA is an immunomodulator, L is a linker, and C is a cell membrane anchor moiety.

206. The method of claim 205, wherein IA is a cytokine.

207. The method of claim 205, wherein the IA is selected from the group consisting of: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof.

208. The method of claim 205, wherein IA is IL-2.

209. The method of claim 205, wherein IA is IL-12.

210. The method of claim 205, wherein IA is IL-15.

211. The method of claim 205, wherein IA is IL-21.

212. The method of any one of claims 205 to 211, wherein L is a CD8a transmembrane-intracellular domain, B7-1 transmembrane domain, B7-2 transmembrane domain, or CD8a transmembrane domain.

213. The method of any one of claims 205 to 211, wherein L is a B7-1 transmembrane domain.

214. The method of any one of claims 205 to 211, wherein L has the amino acid sequence of SEQ ID No. 239.

215. The method of claim 197, wherein the one or more membrane-anchored immunomodulatory fusion proteins are independently according to the formula from N-terminus to C-terminus: S1-IA1-L1-C1-L2-S2-IA2-L3-C2, wherein S1 and S2 are each independently a signal peptide, IA1 and IA2 are each independently an immunomodulator, L1 to L3 are each independently a linker, and C1 and C2 are each independently a cell membrane anchor moiety.

216. The method of claim 215, wherein S1 is the same as S2.

217. The method of claim 215 or 216, wherein C1 is the same as C2.

218. The method of any one of claims 215-217, wherein L2 is a cleavable linker.

219. The method of claim 218, wherein L2 is a furin cleavable linker.

220. The method of any one of claims 215-219, wherein IA1 and IA2 are each independently cytokines.

221. The method of any one of claims 215-219, wherein IA1 and IA2 are each independently selected from: IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFNgamma, TNFa, IFNalpha, IFNbeta, GM-CSF or GCSF or variants thereof.

222. The method of any of claims 215-219, wherein IA1 and IA2 are each independently selected from IL-2 and IL-12, provided that one of IA1 and IA2 is IL-2 and the other is IL-12.

223. The method of any one of claims 215-219, wherein IA1 and IA2 are each independently selected from IL-15 and IL-21, provided that one of IA1 and IA2 is IL-15 and the other is IL-21.

224. The method of any one of claims 215-223, wherein each of C1 and C2 is independently a CD8a transmembrane-intracellular domain, B7-1 transmembrane domain, B7-2 transmembrane domain, or CD8a transmembrane domain.

225. The method of any one of claims 215-223, wherein each of C1 and C2 is a B7-1 transmembrane domain.

226. The method of any one of claims 215-223, wherein each of C1 and C2 has the amino acid sequence of SEQ ID No. 239.

227. The method of any one of claims 197-226, wherein the modified TIL expresses the one or more membrane anchored immunomodulatory fusion proteins under the control of an NFAT promoter.

228. The method of any one of claims 197-227, wherein the modified TIL is transduced with a retroviral vector to express the one or more membrane anchored immunomodulatory fusion proteins.

229. The method of any one of claims 197-227, wherein the modified TIL is transduced with a lentiviral vector to express the one or more membrane anchored immunomodulatory fusion proteins.